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Neuroplasticity of the basal ganglia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease
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Neuroplasticity of the basal ganglia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease
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Content
NEUROPLASTICITY OF THE BASAL GANGLIA IN THE 1-METHYL-4-PHENYL-1,2,3,6-
TETRAHYDROPYRIDINE MOUSE MODEL OF PARKINSON’S DISEASE.
by
Marta Vučković
______________________________________________________________________
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)
December 2010
Copyright 2010 Marta Vučković
ii
ACKNOWLEDGMENTS
I would like to thank my dissertation adviser Dr. Michael Jakowec for welcoming
me to his laboratory in November 2005, and providing me with an opportunity to work on
a very inspirational research project. Throughout the years I spent in his laboratory as a
graduate student, Dr. Jakowec thought me that neuroscience research requires a lot of
patience and perseverance and constantly reminded me that scientific career is full of
challenges, from dealing with revisions of manuscripts for publication, to coming back to
the lab and continuing the work after a good grant proposal have been rejected from a
funding agency. During lab journal clubs Dr. Jakowec showed me how to critically read
and interpret published results, and never take the results of the studies for granted,
even if they come from prestigious scientific journals.
Some of the projects I was involved in during my graduate studies would never
see the light of the day and would not be available for me without the high energy and
drive for new discoveries of Dr. Giselle Petzinger. I am particularly grateful for the
opportunity she has given me to shadow her in the Neurology Clinic at the Keck School
of Medicine, and see first hand how the diagnosis of Parkinson’s disease is made and
what is the standard of care today for patients affected by this disease. This experience
helped me to always keep in my mind the greater goal of our research efforts.
All studies presented in this dissertation are results of collaborative work between
the laboratory of Dr. Jakowec and Dr. Petzinger and a number of other researchers at
University of Southern California. None of the projects presented here would be possible
for me to finish, or be involved in, without significant contribution, help and support from
the following people: Dr. Ruth Wood, Avery Abernathy, M.S., Dr. Angelo Nacca, Dr.
iii
Quanzheng Lee, Dr. Richard Leahy, Dr. Peter Conti, Dr. Rex Moats, Dr. Garnik Akopian,
Dr. John Walsh and Dr. Charlie Meshul.
All mouse behavioral studies presented in Chapter 2 of this dissertation were
preformed in the laboratory of Dr. Ruth Wood from the Department of Cell and
Neurobiology. Her critical guidance and support during these studies were of great value
to me. Avery Abernathy preformed analysis of dopamine concentrations in mouse brain
tissue, presented in Chapters 2 and 3.
Live imaging experiments presented in Chapter 3 were preformed in
collaboration with Dr. Angelo Nacca, from the Department of Radiology, Dr. Quanzheng
Lee and Dr. Richard Leahy from USC School of Engineering, and Dr. Rex Moats from
Saban Research Institute at Children’s Hospital Los Angeles.
Electron microscopy studies presented in Chapter 4 were done in collaboration
with Dr. Charlie Meshul from Oregon Health Sciences University, Portland, Oregon. I
would also like to thank Yi-Hsuan (Lilian) Lai for executing the treadmill exercise
protocol, Elizabeth Hogg for technical help with Golgi-Cox labeling protocol and Avery
Abernathy for his expertise with HPCL dopamine analysis.
Dr. Garnik Akopian preformed electrophysiological experiments presented in
Chapter 5 of this dissertation. Without his help end efforts, that part of the study would
not be possible and would not be presented here. It was a great experience for me to
work closely with Garnik and learn theoretical and experimental principles of
neurophysiology.
Funding support for studies presented in Chapters 2, 3, 4 and 5 were provided by
the Parkinson’s Disease Foundation, Team Parkinson Los Angeles, the Parkinson
Alliance, the Whittier Parkinson’s Disease Education Group, NINDS RO1 NS44327-2 (to
Michael W. Jakowec) and USC CTSI Full Pilot Grant Program (to Giselle M. Petzinger),
iv
and generous gifts from George and MaryLou Boone and Walter and Susan Doniger. I
received the Neuroscience Graduate Program Merit Fellowship for the academic year
2008-2009.
I would like to thank my dissertation committee members for their generous
support during my research projects and their availability for good scientific discussions.
Dr. Ruth Wood helped me the most during my critical first year in the laboratory. Her
guidance in writing the first manuscript was of great value to me. Her guidance
throughout the writing process of this dissertation helped me to finish the manuscript in
timely manner. Her commitment to success of all graduate students makes her a great
mentor and an excellent role model for an academic career. My discussions with Dr.
Carolee Winstein were always very inspirational and I appreciate a lot that she was the
member of my dissertation committee. Collaboration with Dr. John Walsh during my last
research project was fruitful and I am grateful to be given the opportunity to work in his
laboratory and learn principles of electrophysiology. Dr. Janos Peti-Peterdi provided me
with the opportunity to learn principles of multi-photon in vivo imaging and have hands-
on training in advanced microscopy techniques in his laboratory.
I was very fortunate to work with a few very talented young scientists and
graduate students at the beginning of my dissertation research. Among them, Elizabeth
Hogg helped me the most with adjusting to the new laboratory and provided me with
very valuable practical tips of every day work at the bench. Her view on life and
effectiveness at work are truly inspirational. During the time we worked in the lab
together, she became a good friend of mine and helped me finally adjust to the new
culture and new working environment. Working with Jon VanLeuween side by side in the
lab was a great experience for me. His dedication to scientific discovery and always a
great attitude makes him a great role model and a very pleasant person to work with.
v
Lilian Lai, M.S., P.T. helped me with animal training protocols for multiple studies
presented in this dissertation. Lunches and long discussions with Dr. Eleni Antzoulatos
helped me thought the difficult time of my graduate career. Her guidance in writing my
research proposal and dissertation were extremely valuable to me. My deepest thanks
go to my fellow graduate student Kelly Kent. She was there for me in critical moments of
my dissertation writing and her help with editing my writing was extremely important for
me. I would like to thank Dr. Kristina Nowicki for being always there with a helpful
advice, and Raina Pang, Letisha Wyatt and Elizabeth Zuniga for their friendly support in
my efforts to finish my dissertation.
I am very grateful to be given the opportunity to work with a few of very talented
undergraduate students, without whose help my numerous experiments would not be
finished on time. Jason Fuerst and Phil Vitozzi helped during the most critical part of my
dissertation work with animal experiments and data analysis. Both of them will soon
become excellent doctors in their fields of medicine and I am very thankful to them for
taking the time and doing such an excellent job in the laboratory. Alexandra Smith was
one of the first undergraduate students that I had a chance to mentor and teach science.
Along the way, she became my friend outside of the lab, and her energy and
compassion will keep her my friend in the future.
Finally, my deepest thanks go to Dr. Sean Gordon, my husband and my best
friend, without whom I would have not come to Los Angeles and USC to work on my
doctorate. Sean is my greatest role model and a lifelong inspiration, and he reminds me
every day to live my life to the fullest and never give up on my dreams.
vi
TABLE OF CONTENTS
Acknowledgments ii
List of Figures vii
Abstract ix
Chapter 1: Introduction 1
Chapter 2: Memory, Mood, Dopamine, and Serotonin in the 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine-Lesioned Mouse Model of
Basal Ganglia Injury 50 61
Table 2.1: HPLC analysis of dopamine, serotonin, and their
calculated turnover ratios in the dorsal striatum, ventral
striatum, frontal cortex, amygdala, ventral
mesencephalon (VME), and raphe nucleus from
control, 7 days, and 30 days post-MPTP-lesioned mice
(n=5/group) 67 79
Chapter 3: High Intensity Treadmill Exercise Upregulates Striatal
Dopamine D2 Receptor in 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine-lesioned Mice: In vivo PET-Imaging with
[
18
F]fallypride 77 89
Chapter 4: Treadmill Exercise Reverses MPTP Spine Loss in Striatal
Medium Spiny Neurons 94
Chapter 5: Changes in AMPA Receptor Expression and Medium Spiny
Neurons Morphology with Treadmill Exercise in the 1-Methyl -
4-Phenyl-1,2,3,6-Tetrahydropyridine-Lesioned Mouse Model of
Basal Ganglia Injury 108 122
Chapter 6: Conclusions 135
Bibliography 150
Appendix: Additional Publications 181
vii
LIST OF FIGURES
Figure 1.1: Functional organization of the basal ganglia 7
Figure 1.2: Synapses on medium spiny neurons 9
Figure 1.3: Direct and indirect pathway connections in the striatum 44
Figure 2.1: Diagram representing the timeline and order of behavioral
tests following acute MPTP-lesioning (4x20mg/kg, 2h apart) or
saline injections (4x0.1ml, 2h apart) 54
Figure 2.2: Associative memory impairment in MPTP-lesioned mice
measured by the social transmission of food preference test 62
Figure 2.3: Anxiety in mice 7 and 30 days post-MPTP-lesioning measured
in light-dark preference and the hole-board tests 63
Figure 2.4: The effect of acute MPTP-lesioning on depression in mice 7
and 30 days post-treatment measured using sucrose
preference and tail suspension tests 64
Figure 2.5: Acquisition and extinction of fear response in MPTP-lesioned
mice measured in the fear conditioning test 66
Figure 2.6: Dopamine and serotonin levels in MPTP-lesioned mice 68
Figure 3.1: [
18
F]fallypride shows high biding specificity to the mouse
striatum 82 95
Figure 3.2: Exercise improves motor behavior in the MPTP mouse 86
Figure 3.3: Exercise selectively up-regulates DA-D2R but not DA-D1R
striatal protein 87 100
Figure 3.4: Exercise selectively increases [
18
F]fallypride binding potential
(BP) in the striatum of MPTP mice 89
Figure 4.1: High intensity treadmill exercise increases spine density on
striatal MSNs 103 116
Figure 4.2: Electron microscopic analysis of spine area and number of
synapses in dorsal striatum of mice 104
Figure 5.1: Dopamine levels in dorsal striatum of MPTP-lesioned and
control BAC-Drd2-eGFP mice 123
viii
Figure 5.2: Analysis of GluR2 immunoreactivity in direct and indirect
pathway MSNs from striatal tissue sections of BAC-Drd2-
eGFP mice 126 138
Figure 5.3: High intensity treadmill exercise increases spine density on
indirect pathway striatal MSNs in MPTP-lesioned mice 128
Figure 5.4: High intensity treadmill exercise differentially influences type
of spines in striatal MSNs in control and MPTP-lesioned mice 129
Figure 5.5: Treadmill exercise reduces inward rectification of AMPA
receptor mediated excitatory postsynaptic currents on indirect
pathway MSNs in MPTP mice 130
Figure 5.6: Exercise does not influence amplitude or frequency of
sEPSCs in indirect pathway MSNs 131
Figure 5.7: Exercise does not influence the input-output relationship for
corticostriatal synapses on indirect pathway MSNs 132
Figure 6.1: Future directions in studying effects of exercise-induced
synaptic plasticity in striatal pathways of injured basal ganglia 146
Figure 6.2: The big picture: overview of molecular and cellular
mechanisms underlying exercise-induced neuroplasticity in the
basal ganglia based on experimental results presented in this
dissertation 148
ix
ABSTRACT
The mammalian brain is a remarkable organ that continues to fascinate biologists
with its ability to undergo experience-dependent adaptations, and this property is
preserved throughout the life. Brain plasticity on the molecular and cellular level within
neurons is thought to occur in response to neuronal activity, continuously changing the
strength of synapses and thus influencing the connections between neurons. Synapses
are weakened or strengthened in response to specific patterns of neuronal activity. The
underlying mechanism of synaptic plasticity is thought to consist of the regulated release
of neurotransmitters (chemical mediators of neuronal communication) from the
presynaptic side, and changes in the expression levels and availability of corresponding
receptors located predominantly on the postsynaptic side of the synapse. Numerous
studies over the past three decades have focused on elucidating the molecular basis of
synaptic plasticity, with the goal of better understanding the link between transient
changes in neuronal activity in response to experience, and short- and long-term
changes in brain circuitry that underlie learning, memory and adaptive behavior.
However, many key questions remain unresolved. The results of research efforts
presented in this dissertation represent a modest contribution to our increasing
knowledge of experience-dependent brain plasticity and its relevance to human health.
Naturally, the most dramatic changes in the complexity and functionality of any
brain region happen during development, when a single sheet of precursor cells gives
rise to a complex organ with multiple types of neurons, communicating through billions of
connections and using different types of chemical messengers, the neurotransmitters. At
the other end of life, during aging and the neurodegenerative processes, the brain
undergoes a different kind of plasticity, a dynamic process that effectively breaks
connections between neurons, and severely impairs numerous brain functions. Looking
x
from the outside, these two periods of life seem to initiate the most dramatic and highly
dynamic of all the changes in the brain as they are evident through striking changes in
behavior or by the gain or the loss of functions. However, throughout the healthy adult
life, more subtle, yet important plastic changes happen in the brain at both the molecular
and cellular level, enabling animals and humans to learn new motor skills or a new
language and remember places and people they meet. Emerging new research
suggests that dynamics in the brain go beyond learning processes. Accumulating
evidence supports the hypothesis that daily experiences, stress, physical activity, diet,
and lifestyle changes are all factors that cause subtle plastic changes in the brain,
bringing both positive and negative consequences to overall brain function. In this
dissertation, I investigated adult brain plasticity in health and disease. For my research I
used the MPTP mouse model of Parkinson’s disease. This animal model enabled me to
investigate plastic changes in the brain on the molecular and cellular level and also
provided me with a simple model of behavioral changes. The first study of this
dissertation was designed to investigate the plasticity in the serotonin system in different
brain regions following a neurotoxic brain injury resulting in severe loss of dopamine
(presented in Chapter 2). Serotonin is a chemical used by different brain regions for
communication between neurons. Through its signaling in the frontal cortex, amygdala
and midbrain, serotonin controls complex behaviors such as fear response and memory
formation, and its imbalance is closely linked to human psychiatric conditions such as
depression and anxiety. In the study presented in Chapter 2, severe dopamine depletion
caused loss of serotonin in multiple brain regions in the mice treated with MPTP, and
this neurotransmitter imbalance caused memory impairments, but not depression and
anxiety-like behaviors. Three other studies in this dissertation focused on investigating
experience-driven neuroplasticity in the adult mouse brain. Specifically, in Chapter 3, my
xi
studies focused on plastic changes in expression of dopamine receptor D2 in the
striatum of mice lesioned with MPTP and exposed to daily treadmill exercise. Dopamine
receptors are gates of dopamine signaling in the brain and without them the
neurotransmitter cannot be used for communication between neurons. Results
presented in Chapter 3 indicate that daily treadmill exercise for 6 weeks increases
expression of dopamine receptor D2 in the striatum of mice. This finding was also
confirmed using in vivo positron emission tomography (PET) imaging of live mice. Using
the same experimental model in Chapter 4, I examined the plastic changes of medium
spiny neurons (MSNs), the principal neurons in the striatum, focusing on the analysis of
their complex morphology. The analysis showed that 6 weeks of treadmill exercise
increased the spine density on MSNs in the dorsolateral striatum, and this increase was
observed in both control and MPTP-lesioned mice but was more pronounced in the
lesioned mice. This finding was supported by electron microscopy analysis of synapse
number, which showed significant increase in response to treadmill exercise. I
investigated this phenomenon further in Chapter 5 by focusing on a subset of MSNs,
those that are part of the striatal indirect pathway and predominantly express dopamine
D2 receptors. This study used a transgenic mouse model that was engineered to allow
direct visualization of the indirect pathway MSNs in the mouse brain using the
fluorescent microscope. Results of this study showed that spine density changes on
MSNs in response to exercise occur in indirect pathway neurons and future analysis will
investigate if these changes are restricted to this striatal pathway or if they also occur in
direct pathway MSNs. Analysis of glutamate AMPA (alpha-amino-3-hydroxy-5-methyl-4-
isoxazolepropionate) receptor composition in indirect pathway MSNs showed that
treadmill exercise increased expression levels of the GluR2 subunit of this receptor in
both control and MPTP lesioned mice. Electrophysiological recordings support these
xii
findings. Subunit composition of glutamate AMPA receptors is critical for experience-
dependent synaptic plasticity in multiple brain regions, including the striatum. Modulation
of AMPA receptor subunit composition through treadmill exercise training could be a part
of the molecular mechanisms responsible for the benefits of exercise on the brain in both
healthy individuals and people affected with PD.
Taken together, the results of the studies presented in this dissertation contribute
to our knowledge and understanding of adult brain plasticity in health and disease. In
addition, studies in this dissertation contribute to our better understanding of animal
models of human diseases.
1
CHAPTER 1:
INTRODUCTION
The unifying theme of all studies presented in this dissertation is neuroplasticity
mechanisms of the injured brain, and specifically the basal ganglia. Molecular and
cellular processes leading to a neurodegenerative disease, such as Parkinson’s disease
(PD) are very complex and affect not only the basal ganglia nuclei but other brain
regions as well. Despite decades of clinical and basic research, these processes are
still not fully understood. Using a simplified animal model of the disease, studies in this
dissertation attempted to analyze neuroplasticity mechanisms caused by the most
important aspect of PD pathology - dramatic loss of dopamine in the striatum, the main
input nuclei of the basal ganglia system, following death of dopamine producing neurons
in the midbrain. Although the animal model used in the studies robustly captures only
one aspect of PD pathology, results of studies presented here and elsewhere show that
single neurotransmitter depletion from one brain region leads to dysfunction in other
brain systems. In affected patients, the complex processes of neurodegeneration are
reflected in development of a broad array of clinical symptoms, ranging from severe
motor dysfunction to mood disorders and memory impairment. In animal models widely
used in research today, PD pathology causes a limited number of symptoms, with motor
impairments being the most documented in the literature. However, the true value of
animal models of PD lies in the researcher’s ability to examine in detail the complex
processes of neuroplasticity that follow dopamine depletion in the basal ganglia. Our
ability to understand and perhaps promote some of the neuroplasticity mechanisms by
various intervention methods in laboratory animals will ultimately lead to better and
longer lasting therapeutic modalities for patients affected by PD.
2
The goal of this doctoral dissertation was to investigate neuroplasticity of the
basal ganglia nuclei in the MPTP mouse model of PD. The studies presented here focus
on two aspects of the plasticity of the injured basal ganglia: 1) the intrinsic mechanisms
of neuroplasticity following neurotoxin exposure, and 2) behavioral activity-dependent
mechanisms of synaptic plasticity initiated by physical exercise and motor training. The
first part of the thesis (Chapter 2) investigates dynamic changes in levels of the
neurotransmitters dopamine and serotonin throughout the basal ganglia following MPTP
exposure and their relation to the development of non-motor symptoms of PD, such as
anxiety, depression and memory impairments. The second part of the thesis (Chapters
3, 4 and 5) focuses on the molecular and cellular mechanisms by which physical activity
such as treadmill exercise promotes plasticity of dopamine and glutamate
neurotransmission in the dorsal striatum, the main input nucleus of the basal ganglia,
and how this altered communication between neurons improves the function of basal
ganglia circuits following injury. Overall, the main goal of research projects presented in
this dissertation was to understand how changes at the molecular and cellular levels
translate into altered neural circuit function and behavior in an animal model of a
neurodegenerative disease.
1.1. Neuroplasticity
Neuroplasticity refers to the capacity of the nervous system to change its
responsiveness and reactivity as a result of successive activations, or a period of
inactivation. Activation processes could be external (environmental factors, behavioral
activity, neurodegenerative disorders or traumatic brain injury) or could originate from
within the nervous system (developmental cues or pre-programmed gene expression
changes, for example during aging). Neuroplasticity encompasses a broad spectrum of
3
chemical, molecular, cellular and physiological processes within the brain, including
neurotransmitter release, expression of membrane receptors and cellular signaling
molecules, posttranslational modification of proteins, physiological responsiveness of
neurons (neuronal excitability), long-term potentiation and long-term depression of
synaptic connectivity, structural plasticity (dendritic tree branching, the length of the tree,
spine density, diameter of dendrites), change in the number of synapses and changes in
network connectivity.
A more narrow term of synaptic plasticity refers to the ability of individual neurons
to change the strength of communication between one another. Dynamic change in
strength of neuronal communication modulates activity of brain circuits and ultimately
influences behavior. Modulation of the synaptic strength can be initiated by a variety of
factors, including developmental cues, stress, hormones, behavioral activity, learning
and memory formation. In the current literature, the process of synaptic strength
modulation in response to behavioral activity is commonly called experience-dependent
synaptic plasticity and it is the focus of chapters 3, 4 and 5 of this dissertation.
D.O. Hebb first introduced the term synaptic plasticity in 1949. He postulated that
when two neurons are communicating with each other via an active synapse, the
strength of their connection grows stronger if both neurons are repeatedly firing at the
same time (Hebb 1949). It is important to note that synaptic plasticity refers to the
opposite process as well: if the two neurons connected via a synapse are inhibited from
firing at the same time, the strength of synaptic transmission weakens. The first
experimental evidence to support Hebb’s theory came in 1973 when researchers
showed that persistent activation of excitatory synapses in the rabbit hippocampus leads
to a long lasting increase in the neurotransmission that can last for hours and days (Bliss
& Gardner-Medwin 1973; Bliss & Lomo 1973). Today, this phenomenon of long-lasting
4
increases in synaptic efficacy is commonly called Long-Term Potentiation (LTP) and the
opposite process of long lasting weakening of synaptic transmission is referred to as
Long-Term Depression (LTD).
Synaptic plasticity is driven by changes in molecular composition of synapses
(Citri & Malenka 2008). The synapse is a complex and highly specialized sub-cellular
structure composed of a presynaptic and postsynaptic membrane originating from two
neurons, and the synaptic cleft in between. The presynaptic side is the source of
neurotransmitters, released in packages of synaptic vesicles in response to a stimulus.
The release of neurotransmitters is highly regulated under physiological conditions, but
at the same time can be modulated in response to the activity of the presynaptic neuron.
On the postsynaptic side, the critical units that facilitate synaptic transmission are
neurotransmitter receptors and their auxiliary molecules involved in anchoring to and
removal from the active zone, as well as intracellular signaling cascades. The
expression levels and availability of receptors are regulated in part by neurotransmitter
presence in the synaptic cleft and in part by the physiological state of the postsynaptic
neuron. During the course of a neurological disease, or following a neurotoxic injury, any
pathological change in the synaptic components (loss of the presynaptic side or a whole
neuron, loss of, or excess in release of a neurotransmitter, or loss of the postsynaptic
side) results in dramatic change of synaptic strength or loss of the synapse altogether.
On the other hand, under physiological conditions, behavioral activity and/or response to
a novel environment triggers changes in excitability of either the pre-or post-synaptic
neuron or both, which in turn modify synaptic strength.
Accumulating evidence from both clinical and basic science research suggest
that activity-dependent synaptic plasticity is crucial for normal functioning and learning
processes in all brain regions, from the cerebral cortex (Feldman 2009), and the basal
5
ganglia (Kreitzer & Malenka 2008), to the cerebellum (Hansel et al 2006). Although
expression of synaptic plasticity in various brain structures differs depending on the type
of neurons involved, results from numerous studies suggest that basic molecular
mechanisms involved in different forms of synaptic plasticity are shared between
different brain regions.
Studies in this dissertation were designed to investigate molecular and cellular
correlates of activity-dependent synaptic plasticity in the basal ganglia in a mouse model
of PD. In order to explain the rationale for the studies in this dissertation, it is important
to begin with a discussion of the basal ganglia components and neuroplasticity
mechanisms underlying normal basal ganglia function that support learning a new motor
task, as well as pathological changes leading to motor dysfunction seen in PD.
1.2. Anatomy and function of the basal ganglia
The basal ganglia are a group of interconnected sub-cortical nuclei involved in
motor control, motivation, procedural learning, and cognitive and limbic function. The five
nuclei of the basal ganglia include the striatum, the globus pallidus (external and
internal), the subthalamic nucleus, and the substantia nigra (Fig 1.1). The striatum is the
main input nucleus of the basal ganglia and a major target of dopamine projections from
the substantia nigra. It consists of a system of interconnected neurons that receive
topographically organized excitatory glutamate inputs originating from the cerebral
cortex or the thalamus with dense modulatory dopamine innervations from the midbrain
(Smith et al 2009a). The striatum is a major site of synaptic plasticity in the basal ganglia
(Bolam JP 2000; Gerdeman et al 2003; Gerfen 2000). After processing in the striatum,
information is sent via GABA neurotransmission to the external globus pallidus (GPe), or
to the output nuclei of the basal ganglia, the internal globus pallidus (GPi) and the
6
substantia nigra pars reticulata (SNpr), which in turn project to the thalamus and brain
stem, forming the corticostriatal-thalamic loop (Parent & Hazrati 1995; Smith et al
1998b).
7
Figure 1.1: Functional organization of the basal ganglia. Diagram showing the nuclei of
the basal ganglia and their connections to the cortex, thalamus, substantia nigra pars
compacta (SNpc) and pars reticulata (SNpr). Major neurotransmitters of the basal
ganglia are: Glu - glutamate, DA - dopamine, Ach - acetylcholine, GABA - gamma-
aminobuteric acid.
8
The majority of cells in the striatum release the inhibitory neurotransmitter γ-
amino-butyric acid (GABA), including the large population of principal neurons and a
smaller population of interneurons. In addition, the striatum contains a small number of
giant cholinergic interneurons which can be distinguished from other cells by their large
cell bodies, tonic activity in vivo and dense axonal arborization (Zhou et al 2002). The
principal neuronal type in the striatum is the medium spiny neuron (MSN), characterized
by cell body diameter of about 15-18µm, high dendritic spine density, negative resting
potential and slow firing rates in vivo. Each MSN has a very large number of spines.
Average spine density per neurons in a healthy adult male mouse is in the range of 8-10
spines per 10 micrometers of dendritic length. Dendritic spines are the sites of diverse
synaptic plasticity events, ranging from changes in synaptic strength (Sorra & Harris
2000; Yuste & Bonhoeffer 2001) to experience-dependent plasticity in living animals
(Zito & Svoboda 2002). Striatal MSNs receive two types of inputs through their dendritic
spines – excitatory glutamate neurotransmission originating from the motor cortex or the
thalamus, and modulatory dopamine neurotransmission originating from the SNpc (Dube
et al 1988; McGeorge & Faull 1989; Smith et al 1994). Due to their close anatomical
position on dendrites of MSNs, dopamine and glutamate terminals influence
neurotransmission from each other’s terminals in addition to controlling the release of
their own neurotransmitters (Bamford et al 2004a; Bamford et al 2004b; Morari 1996;
Morari et al 1994; Yamamoto 1992; Zhang & Sulzer 2003) (Figure 1.2).
9
Figure 1.2: Synapses on medium spiny neurons. Excitatory glutamate afferent
(corticostriatal neuron) makes a synapse on the spine head of the medium spiny neuron,
and dopamine-producing (nigrostriatal) neuron forms synapse on the spine neck.
Complex array of intracellular signaling cascades inside of spine head couples
dopamine and glutamate neurotransmission at the molecular level. Role of the brain-
derived neurotrophic factor (BDNF) signaling through its receptor TrkB is important for
activity-dependent synaptic plasticity. DA – dopamine, Glu – glutamate, DR – dopamine
receptor on MSN, DR2 – dopamine D2 receptor on the presynaptic glutamate terminal,
AMPAR and NMDAR – glutamate receptors on MSN, GluR2 – subunit of glutamate
AMPA receptors critical for experience-dependent synaptic plasticity,
Electron microscope studies have observed that the synapses between
dopamine axons and the dendrites of MSNs are precisely organized. The dopamine
axon synapses on the dendritic spine neck while the glutamate axon synapses on the
spine head. This synaptic architecture suggests that one of the functions of dopamine is
to modulate the effects of excitatory corticostriatal glutamate drive on MSNs (Deutch et
al 2007).
10
The most influential model of basal ganglia circuitry is based on anatomical and
functional segregation of information processing in the striatum into the direct and
indirect pathway, each of them acting in an opposing way to control movement (Albin et
al 1989; Alexander & Crutcher 1990; DeLong 1990). These two pathways run in parallel
through the striatum, make circuit loops connecting the motor cortex, basal ganglia
nuclei and the thalamus and are differentially modulated by dopamine. The direct
pathway (involving medium spiny neurons that contain substance P/dynorphin, and
predominantly express dopamine D1 receptors and muscarinic M4 receptors) connects
the striatum to GPi and SNpr, while the indirect pathway (involving MSNs containing
enkephalin and expressing predominantly dopamine D2 receptors and adenosine A2A
receptors) connects the striatum to GPe and STN (Gerfen et al 1990; Smith et al 1998a).
Both types of MSNs integrate a large number of inputs to generate spiking activity as the
only output from the striatum to the rest of the basal ganglia nuclei. Thus, neuronal
plasticity in the striatum directly regulates the outflow of information from the basal
ganglia to the thalamus and the cortex, modulating motor control and learning. The
output nuclei of the basal ganglia are GPi and SNpr (Figure 1.1).
The effects of the direct and indirect pathways on excitatory thalamo-cortical
projections are contrasting - the direct pathway causes disinhibition of target neurons
leading to activation of cortical premotor circuits and facilitation of movement, while the
indirect pathway has an inhibitory effect which reduces cortical premotor drive and
inhibits movement. An important component of this simplified model is dopamine
regulation of MSNs firing rate. Dopamine receptors belong to the family of G-protein
coupled receptors and have seven transmembrane domains. Their activation modulates
activity of other neurotransmitter receptors on MSNs, such as glutamate receptors, via
intracellular signaling cascades. Through activity of the D1 receptor coupled with a G
s
11
regulatory subunit, dopamine facilitates MSN output, while signaling on D2 receptors
coupled with a G
i
subunit inhibits activation of MSNs (Surmeier et al 2007). Interestingly,
while dopamine signaling regulates the activity of direct and indirect pathway in opposing
ways, ultimately it enhances movement (Albin et al 1989), as seen when severe
dopamine loss in PD causes akinesia.
In PD, loss of dopamine producing neurons in the midbrain causes degeneration
of dopamine projections from SNpc to the striatum. However, the striatum is not the only
brain region affected by dopamine loss. Modulatory effects of dopamine on the frontal
cortex, hippocampus and limbic system are progressively lost during the course of the
disease (Ressler & Nemeroff 2000; Walsh & Bennett 2001). In addition, other
neurotransmitter systems, such as acetylcholine and serotonin are affected in PD
patients. Studies in Chapter 2 of this dissertation investigated dopamine and serotonin
dysfunction in key brain regions, such as dorsal and ventral striatum, amygdala and the
prefrontal cortex in the MPTP mouse model of PD, and their relationship to the
development of non-motor symptoms such as depression, anxiety and memory
impairment. The results of these studies indicate that loss of dopamine producing
neurons in the midbrain leads to severe serotonin loss throughout the brain and
progressive development of non-motor symptoms in the MPTP mouse.
The classical model of PD postulates that dopamine loss in the striatum
increases the activity of direct pathway MSNs and decreases the activity of the indirect
pathway MSNs. Together these changes lead to increased GABA inhibition of the
thalamus by the output nuclei of the basal ganglia (Albin et al 1989; DeLong 1990;
DeLong & Wichmann 2007; Graybiel et al 2000). However, this model oversimplifies the
role of dopamine in basal ganglia function (Nicola et al 2000). Loss of dopamine
signaling influences long-term synaptic plasticity of cortical glutamate projections to the
12
striatum and activation of MSNs, and triggers substantial secondary morphological
changes, such as dendritic spine loss on MSNs (Calabresi et al 2007; Raju et al 2008;
Smith et al 2009b; Villalba et al 2009). Studies in Chapters 4 and 5 of this dissertation
investigated activity-dependent morphological changes on MSNs (spine density) along
both the direct and indirect pathways, as well as their relationship to neuronal
excitability.
1.3. Synaptic plasticity of the basal ganglia in healthy and dopamine-depleted
brain
The output from the striatum to the rest of the basal ganglia nuclei depends upon
complex interactions between the intrinsic properties of MSNs and their excitatory and
inhibitory synaptic inputs from the cortex and thalamus. Dopamine action as a
neuromodulator alters both cellular and synaptic function of MSNs to adjust output
through the direct and indirect pathways and in this way regulates motor function. The
following paragraphs discuss the present knowledge and proposed mechanisms of
dopamine activity on synaptic plasticity in the striatum, in healthy and injured basal
ganglia.
In addition to other brain regions, the striatum is a site of activity-dependent
neuroplasticity (Kreitzer & Malenka 2008). Two neurotransmitter systems influence the
activity of MSNs in direct and indirect pathways - fast excitatory glutamate
neurotransmission from the cortex and thalamus and slow dopamine signaling from the
midbrain. In addition to these two regulatory systems that originate from extra-striatal
brain regions, inhibitory GABA neurotransmission is also involved in synaptic plasticity of
MSNs and is generated within the striatum. Activity-dependent synaptic plasticity
influences neuronal excitability throughout the basal ganglia and it has a crucial role in
13
adaptive motor control and procedural learning. Synaptic plasticity is also responsible for
pathologically altered circuits seen in neurodegenerative diseases, such as PD.
Studies of excitatory synapses on MSNs using electrophysiological recordings
and paired-pulse stimulation in the healthy mouse striatum revealed that cortical and
thalamic afferents making synapses onto indirect pathway neurons have higher
probability of glutamate release compared to those connecting to direct pathway
neurons (Ding et al 2008; Kreitzer & Malenka 2007; Mallet et al 2006). Higher probability
of glutamate release suggests that excitatory synapses on indirect pathway MSNs are
more efficient in stimulating target neurons. This conclusion is in agreement with
observed high firing rates on indirect pathway MSNs in vivo (Mallet et al 2006).
However, similar studies that investigated properties of excitatory glutamate synapses
on striatal MSNs by positioning the stimulating electrode outside of the striatum, showed
no difference in release probabilities between the two types of MSNs (Ding et al 2008).
This observation indicates that different sets of cortical or thalamic afferents differentially
stimulate neurons from both striatal pathways, with a possibility that some afferents
stimulate their targets stronger than others. The above mentioned paired-pulse ratio
technique is based on the property of synapses to release only readily available packets
of neurotransmitters from the presynaptic terminal upon electrical stimulation. This
protocol involves two consecutive stimulation pulses to the target tissue, separated by a
brief period of time. The first stimulation pulse mobilizes the readily available pool of
neurotransmitter vesicles, producing a strong response. The second electrical pulse
causes a smaller synaptic response, as the rest of the neurotransmitter vesicles are
being released. The ratio of the presynaptic response from two stimulating pulses is
calculated to measure the probability of a neurotransmitter release. A lower paired pulse
ratio is indicative of a higher probability of neurotransmitter release, while a higher ratio
14
indicates a low probability of release. This electrophysiological technique was used to
investigate striatal synaptic plasticity of indirect pathway MSNs following MPTP lesion
and high intensity treadmill exercise in Chapter 5 of this dissertation.
Dopamine is a modulator of excitatory glutamate neurotransmission in the
striatum. MSNs express dopamine receptors D1 and D2 on dendrites and dendritic
spines that make excitatory synaptic connections with cortical and thalamic axons. In
addition, dopamine activity on D2 receptors expressed on presynaptic corticostriatal and
thalamostriatal terminals modulates glutamate signaling through mechanisms of
glutamate receptor vesicle cycling (Bamford et al 2004a; Bamford et al 2008). However,
molecular mechanisms of this effect of dopamine on presynaptic glutamate release are
not fully understood and may involve the action of endocanabinoids released from MSNs
in the dorsal striatum that have been shown to directly act on presynaptic
neurotransmitter release (Giuffrida et al., 1999; Kreitzer and Malenka, 2005; Yin and
Lovinger, 2006).
Experimental high-frequency stimulation (HFS) protocols in striatal slice cultures
in vitro lead to long lasting synaptic plasticity in the form of the long-term depression
(LTD) of the synaptic strength and reduction of glutamate release (Calabresi et al 1992;
Choi & Lovinger 1997; Lovinger et al 1993; Walsh 1993). A proposed mechanism for this
observation involves activation of both dopamine and glutamate receptors at the
excitatory synapses on MSNs. This type of synaptic plasticity has been documented on
both types of striatal MSNs, from both direct and indirect pathways in healthy animals.
Additional studies have shown that signaling molecules required for maintenance of
striatal LTD include dopamine D2 receptors, type I metabotropic glutamate receptors
(mGluRs) coupled to small G proteins (Gq subunit), voltage gated L-type calcium
15
channels, and endocanabinoid CB1 receptors (Calabresi et al 1992; Calabresi et al
1997a; Choi & Lovinger 1997; Gerdeman & Lovinger 2003; Kreitzer & Malenka 2005;
Sung et al 2001). The basic model proposes that HFS induces glutamate release and
subsequent activation of mGluRs on the postsynaptic side, as well as dopamine release
and activation of D2 receptors. In parallel, MSNs depolarization activates L-type calcium
channels. The proposed mechanism also involves endocanabinoid release from MSNs,
targeting CB1 receptors on the presynaptic membrane, and induction of LTD (Kreitzer &
Malenka 2008). Other molecules implicated in this type of synaptic plasticity include
dopamine D1 receptors, DARPP-32 and nitric oxide released from interneurons
(Calabresi et al 1999; Calabresi et al 2000).
Another form of synaptic plasticity, long-term potentiation (LTP) has also been
reported in the striatum, however, the molecular mechanisms of its induction and
maintenance are not well understood (Calabresi et al 2000; Kerr & Wickens 2001).
Activation of dopamine D1 and glutamate NMDA receptors and signaling through protein
kinase A seems to be involved in striatal LTP (Centonze et al 2003; Kerr & Wickens
2001).
In addition to principal neurons, cortical and thalamic excitatory inputs also
innervate large numbers of other cell types in the striatum, including GABAergic and
cholinergic interneurons (Koos & Tepper 1999; Parent & Parent 2006), although
molecular mechanisms of synaptic plasticity at these synapses has not been studied in
detail. In addition, different classes of GABAergic interneurons form inhibitory synapses
on direct and indirect pathway MSNs (Koos et al 2004; Kubota & Kawaguchi 2000;
Narushima et al 2006). Their activity is also modulated by dopamine (Delgado et al
2000). The number of GABA interneurons in the striatum is small in comparison with
16
other cell types, their fast spiking capability makes them rapidly and easily responsive to
excitatory glutamate inputs and they can significantly contribute to activity-dependent
synaptic plasticity in the striatum (Fino et al 2008; Mallet et al 2005; Plotkin et al 2005).
Additional complexity to the above discussed synaptic plasticity models
originates from collateral connections identified between a subset of striatal MSNs
(Somogyi et al 1981; Wilson & Groves 1980). These connections are weak and difficult
to detect physiologically (Jaeger et al 1994). Direct synapses between MSNs are
predominantly unidirectional, form on distal dendrites, and show higher failure rates
compared to synapses from fast spiking interneurons (Koos et al 2004; Taverna et al
2004; Tunstall et al 2002). Specificity of collateral connections is complex: direct
pathway MSNs preferentially synapse on other direct pathway MSNs, whereas indirect
pathway MSNs appear to innervate both subtypes equally (Taverna et al 2008).
Translated to functional outcome, this connection pattern suggests that increased
indirect pathway activity (correlated with inhibition of movement) can influence direct
pathway output. Effects of dopamine on MSN lateral connections are mediated through
receptor activity, D1 receptor activation leads to excitation while D2 receptor activation
inhibits collateral connections (Guzman et al 2003; Tecuapetla et al 2007).
Loss of dopamine in PD and animal models of the disease causes compensatory
changes on the neurons in the striatum. Studies in human PD patients (Obeso et al
2000) and animal models of PD (Filion & Tremblay 1991; Filion et al 1991; Mallet et al
2006) suggest that striatal dopamine depletion leads to an imbalance in striatal circuits,
resulting in increased activity of indirect pathway MSNs and decreased direct pathway
output. This pathological modulation of striatal circuits leads to decreased activity in GPe
and increase activity in GPi. In addition, numerous cellular and synaptic changes occur
17
in response to dopamine loss, affecting both the direct and indirect striatal pathways
(Albin et al 1989; DeLong 1990). One of the early compensatory changes is increased
activity of dopamine receptor D1 and its downstream signaling cascades (Mishra et al
1974). Additional changes include increased MSN membrane resistance (Fino et al
2008) and spine loss on indirect pathway MSNs (Day et al 2006). Functionally,
dopamine depletion causes the loss of indirect pathway LTD, causing these neurons to
express LTP when stimulated in vitro (Kreitzer & Malenka 2007; Shen et al 2008).
Additionally, new evidence suggests that following dopamine depletion, direct pathway
MSNs exhibit LTD instead of LTP (Shen et al 2008). Together, these observations are in
agreement with the general model of PD: dopamine depletion causes hyper-activity of
the indirect pathway MSNs and hypo-activity of the neurons in the direct pathway.
Taking into account complex neuroplasticity mechanisms in the basal ganglia
triggered by dopamine depletion, and the activity-dependent synaptic plasticity
mechanisms that have the potential to modulate basal ganglia activity in conditions such
as PD, it is of great importance to further study these processes in animal models in
order to develop new therapies and treatments for patients affected by PD. Studies
presented in this dissertation are part of current research efforts to better understand
synaptic plasticity in PD and cellular and molecular processes underlying activity-
dependent plasticity in the basal ganglia of MPTP mouse model.
1.4. Experience dependent brain plasticity
The term “behavioral plasticity” coined by William James in 1895 refers to the
ability of human behavior to carry out meaningful change (Cotman & Berchtoldb 2002).
Since then, the concept of plasticity has been developed to include changes in the brain
at the molecular, cellular and network levels and today it refers to “adaptive changes in
18
brain organization brought about by behavioral experience” (Black et al 1990).
Behavioral stimulation and activity influence brain health and ultimately support
behavioral plasticity (Cotman & Berchtold 2002).
Another process called “activity-dependent neuroplasticity” refers to modifications
within the central nervous system (CNS) in response to physical activity that has an
ultimate goal of learning a new skill (Adkins et al 2006). This definition originates from
research of motor cortex plasticity, an extensively studied process, where motor training
induces experience-specific patterns of plasticity across the motor cortex and the spinal
cord.
Many aspects of behavioral experience influence synaptic connectivity (Bailey &
Chen 1988; Greenough et al 1978; Greenough et al 1973). In order to discuss this
process in detail, it is necessary to define experience dependent brain plasticity. When
talking about the category of “behavioral experience” many examples emerge as
relevant for molecular, cellular and functional brain plasticity, such as brain development,
learning, physical activity or exercise, stress, emotional experience, acute or chronic
neurodegenerative diseases, brain injury or trauma, sensory deprivation, motor
deprivation (paralysis) and others. As is relevant for studies in this dissertation,
behavioral experience encompasses physical activity (exercise), learning of new motor
skills and disability originating from a chronic brain disease. Chronic diseases of the
brain, such as Alzheimer’s disease and Parkinson’s disease cause dysfunction and
death of neurons, leading to decreased neurotransmitter levels throughout the human
brain and functional decline. On the other hand, animal research studies have shown
that voluntary and forced motorized treadmill exercise as well as forced use of an
impaired limb cause synaptic plasticity in the striatum, motor cortex and the
19
hippocampus, respectively (Black et al 1990; Petzinger et al 2010; Xu et al 2009; Yang
et al 2009).
One of the biggest and the most important questions in the field today is whether
the above mentioned maladaptive brain plasticity processes are reversible and if so, to
what extent? Furthermore, the question remains whether these processes have common
molecular mechanisms. Current understanding based on clinical observations is that all
chronic diseases of the brain have a time window of reversible synaptic decline, e.g. as
degeneration processes develop, changes are potentially reversible, until the capacity
for compensation is exhausted, and pathological processes lead to irreversible damage.
1.5. Cellular and molecular mechanisms of experience dependent brain plasticity
Synaptic plasticity shapes neuronal output through modifications of signaling
molecules inside of a neuron. Dendritic spines and synapses, small protrusions of
postsynaptic cell membrane forming highly specialized cellular compartments whose
function is to support synaptic transmission, are the sites of these dynamic changes.
Due to their shape and size, dendritic spines are suitable as unique microenvironments
for ions (such as Ca
2+
) and enzymes (such as protein kinases) important for sustaining
molecular processes underlying synaptic transmission and plasticity. Dendritic spines act
as Ca
2+
compartments. Due to their small volume and lack of mitochondria, spines
accumulate high Ca
2+
concentrations, crucially important for phosphorylation of proteins
involved in synaptic plasticity. For example, transient elevation of Ca
2+
concentration is
important for the induction of LTP (Segal 2005).
Experience dependent synaptic plasticity initiates growth, retraction, or structural
reorganization of existing and new spines and synapses. It is hypothesized that in the
hippocampus, plastic changes in shape and size of dendritic spines is a cellular
20
substrate for memory formation and storage. Numerous studies have shown that in the
hippocampus, induction of LTP causes changes in spine density (Segal 2005).
Since the first description of the dendritic spine by Ramon Y Cajal in 1899 (Cajal
SR, 1995, Histology of the Nervous System of Man and Vertebrate; first published
1899), the idea that the spine is a locus of long-term synaptic plasticity and memory
formation has been preoccupying many neuroscientists. This hypothesis is supported by
observations that enriched environments and learning of new skills contributes to spine
formation (Black et al 1990; Rosenzweig & Bennett 1996; Xu et al 2009; Yang et al
2009).
Dendritic spine structure serves to aid the efficient function of neurotransmission.
Each spine consists of a head region connected to the parent dendrite by a neck. The
head and the neck can have variable diameter or length, giving rise to a variety of spine
types. The most common type is the mushroom spine, with a large head and thin neck.
Then there are thin spines with a small head and very long neck. Another form of spine
is stubby, having a large head with no obvious neck. During development, mature spines
develop from dendritic fillopodia, long and thin processes extending from the dendrite.
Fillopodia can develop into a mature spine upon contact with an axon terminal.
Different shapes and sizes of spines are linked to different functions. Thin spines
are considered to be morphologically unstable and not efficient in synaptic transmission.
With their small heads contributing smaller postsynaptic membrane surface area and
consequently small volumes at the postsynaptic side of the synapse, thin spines are not
well suited for high efficiency of neurotransmission and activity-dependent Ca
2+
influx.
The stubby spines are considered to be mature and stable. Mushroom spines have
stable architecture and are considered to be the site of memory storage in hippocampal
neurons (Bourne & Harris 2007). In addition, mushroom spines support rapid elevation
21
of local Ca
2+
concentration due to the limited diffusion rate out from the spine area
through their thin neck. The role of different shapes and sizes of spines and their
relationship to spine function is not completely understood, and this issue is the focus of
numerous discussions in the current literature. Studies in Chapters 4 and 5 of this
dissertation investigated experience-dependent plasticity of spine morphology on MSNs
in the dorsal striatum of MPTP mice. Presently, there is a general agreement among
researchers that large spines produce large synaptic responses (Matsuzaki et al 2001).
This supports the hypothesis that larger spines with large heads form synapses with
more glutamate receptors and their interacting proteins, producing a large synaptic
response upon activation. Also, there is a strong correlation between the spine head size
and Ca
2+
concentration inside of the spine during synaptic transmission. However,
numerous studies of synaptic transmission in neurons with genetically overexpressed or
deleted key proteins involved in maintenance of spine structure have reported marked
changes in spine morphology without significant changes in miniature excitatory
postsynaptic currents (mEPSC) (Boda et al 2004; El-Husseini et al 2000; Pilpel & Segal
2004). The mEPSC is the electrophysiological signature of the strength of synaptic
transmission through the dendritic spine. Recording mEPSCs makes it possible to
capture a single excitatory synaptic event on a given spine. During recordings, an
amplitude increase of a mEPSC indicates a larger synaptic transmission through a given
synapse, and an increase in frequency indicates the presence of larger number of
synapses on a neuron. For this reason, recordings of mEPSC are well established as
means of directly measuring the synaptic response on a single synapse. This
electrophysiological method was used in studies presented in Chapter 5 of this
dissertation to investigate activity-dependent synaptic plasticity of MSNs from the
indirect pathway in the dorsal striatum of MPTP lesioned mice.
22
One consistent observation reported by several independent research groups is
that spine heads undergo rapid expansion following a tetanic stimulation of hippocampal
neurons in culture or in brain slices (Segal 2005). On the other hand, multiple studies
have confirmed the existence of morphological changes on striatal MSNs in response to
dopamine depletion, resulting in increased synaptic efficacy in glutamate
neurotransmission in the striatum. The observed morphological changes include
increased volume of dendritic spines and presynaptic terminals (Meshul et al 2000;
Meshul et al 1999). The morphology of mature mushroom spines is shaped by actin
cytoskeletal building blocks in response to neurotransmitter-evoked synaptic activity and
growth factor signaling, such as BDNF. On the molecular level, actin polymerization and
de-polymerization is controlled by a group of small GTPases (such as Cdc42, Rac1 and
RhoA) that activate protein kinases involved in actin phosphorylation (Saneyoshi et al
2009). From the studies performed in cultured hippocampal neurons and later replicated
in cultured hippocampal slices, it is now clear that plasticity of spine morphology is
regulated by a complex network of signaling cascades. At excitatory glutamate
synapses, Ca
2+
entry through NMDA receptors triggers a sequence of biochemical
signaling cascades. Elevated Ca
2+
concentration inside of dendritic spines activates
calcium calmodulin-dependent protein kinases (CaMKs). The family of CaMKs, together
with small GTPases and protein kinases such as PAK1/3 regulate phosphorylation of
actin and cytoskeleton re-organization leading to changes in spine morphology.
Spine heads are the location of excitatory synapses, dynamic functional units of
neurons, which are able to modulate their strength in response to external stimuli.
Receptor trafficking within the spine can be very rapid, (this seems to be particularly true
for glutamate AMPA receptors), and this mechanism allows for fast changes in
neurotransmission in response to external stimuli (Man et al 2000). Likewise, plasticity of
23
spine morphology reflects the functional state of the synapse and functional plasticity of
neurons, and together, these processes influence the function of larger brain regions.
This hypothesis has been at the center of intense research efforts over the past decade,
with a particular interest in spine plasticity at hippocampal neurons and its relationship to
memory formation and storage (Segal 2005).
There are two indentified cellular mechanisms of spine formation (Segal et al
2000). De novo formation of spines is the predominant mechanism during neuronal
development. On the other hand, formation of spines from excited synapses on dendritic
shafts has been observed in adult neurons. In development, the immature spine, or
filopodium, shoots off from the parent dendrite by means of rapid polymerization of the
actin cytoskeleton, forms a synapse with a presynaptic neuron and then collapses to
form a “mature spine”, which has a 1-2µm length and a defined spine head at the site of
synapse formation. This process involves a variety of adhesion molecules and
accumulation of a postsynaptic density protein of 95kDa (PDS-95), the major building
block of the postsynaptic density complex, at the spine head (Abe et al 2004; El-
Husseini et al 2000). Mature neurons have few fillopodia, indicating that spine formation
in adulthood is supported by different mechanisms than during development (Dailey &
Smith 1996). Spine pruning is a frequent process in adult neurons and it seems to be a
part of normal synaptic plasticity. In contrast to spine formation that is associated with
long-term potentiation (LTP), it is thought that spine pruning is associated with long-term
depression (LTD), however more evidence is needed to confirm this hypothesis.
Local protein synthesis is an important mediator of synaptic plasticity and spine
formation. It is regulated by locally synthesized translation factors, initiated by synaptic
activity. Target proteins are translated from selected mRNA, transported into dendrites
from the nucleus (Costa-Mattioli et al 2009). In addition, an alternative mechanism of
24
translational control was recently discovered in developing neurons. It relies on miRNA,
small non-coding transcripts of ribonucleic acids whose function is to interfere with local
protein synthesis by destabilizing or suppressing translation of specific mRNA molecules
(Klein et al 2005; Schratt 2009). This process involves RNAase activity of enzyme Dicer
which is found in spines and whose activity can be triggered by elevated Ca
2+
concentrations.
Neurotrophins in general, and brain derived neurotrophic factor (BDNF) in
particular, are considered good candidates for molecules mediating the activity-
dependent structural plasticity of dendritic spines. Research of dendritic plasticity in
cerebral cortex, cerebellum and hippocampus has shown that BDNF signaling through
its receptor TrkB and mitogen activated protein kinase (MAP kinase) signaling pathway
modulates the complexity of dendritic architecture (Danzer et al 2002; McAllister et al
1995; Mertz et al 2000; Tolwani et al 2002; Tyler et al 2002; Yacoubian & Lo 2000). The
extracellular signal-regulated protein kinase (ERK) is a candidate molecule downstream
of TrkB receptor that is thought to mediate dendritic spine growth in response to BDNF
signaling. The phosphorilated ERK1/2 translocates to the nucleus where it activates
nuclear signaling via cAMP response element-binding protein (CREB) transcription
factor that upregulates expression of many target genes. Evidence to support this
hypothesis comes from studies that showed ERK1/2 signaling to be involved in synaptic
plasticity, memory formation, long term stability of dendritic fillopodia on hippocampal
neurons and increases in spine density on hippocampal CA1 neurons (Alonso et al
2004; Blanquet 2000; Finkbeiner et al 1997; Gottschalk et al 1999; Pizzorusso et al
2000).
25
1.6. Introduction to the studies in this dissertation
Studies presented in Chapter 2 investigate neurochemical changes in the basal
ganglia and brain regions involved in affective behaviors, fear, and memory formation.
These regions are also targets of midbrain dopamine projections. In concert with
measurements of dopamine and serotonin, studies in this chapter also analyzed
affective behavior and memory in mice treated with MPTP, 7 and 30 days after
neurotoxin exposure. Results of these studies show that loss of dopamine cells in the
midbrain causes significant depletion of this neurotransmitter in brain areas involved in
mood and memory. In addition, serotonin levels were significantly lower in the striatum,
amygdala, and frontal cortex in MPTP treated mice compared to controls. Low levels of
dopamine and serotonin led to increased fear response and associative memory
impairments in MPTP-lesioned mice, evident 30 days following neurotoxin exposure.
The timeline of observed affective behaviors indicate that there is a threshold of
neurotransmitter loss in multiple brain regions that once crossed, leads to development
of non-motor symptoms in PD.
The goal of Chapters 3, 4 and 5 is to understand the molecular mechanisms by
which physical activity such as treadmill exercise modulates neurotransmission of the
injured basal ganglia and ameliorates its dysfunction in the MPTP mouse model of PD.
These studies test the hypothesis that behavioral activity such as high-intensity treadmill
exercise stimulates and maintains neuronal input from the motor cortex into the striatum
and the basal ganglia. This frequent upregulation of neuronal activity can initiate
synaptic plasticity and protect striatal neurons from dysfunction following neuronal injury
and dopamine loss. Studies examining exercise-induced modulations of the injured
basal ganglia in animals provide a foundation for designing new intervention programs
involving treadmill running with the ultimate goal of slowing PD progression.
26
Studies presented in Chapter 3 sought to examine the effects of exercise on
regulation of dopamine D1 and D2 receptors in MPTP-lesioned mice in an effort to
determine the extent to which receptor-mediated mechanisms may be involved in
exercise-induced synaptic plasticity. Dopamine D2 receptor ligand [
18
F]fallypride and in
vivo PET imaging was used to investigate the effects of treadmill exercise on the binding
potential of D2 receptors in the striatum of MPTP-lesioned mice. In addition, western
immunoblot analysis was used to examine expression levels of the D1 and D2 receptor
proteins in the same brain region. The results show that while high-intensity treadmill
exercise upregulated D2 receptors in the dorsal striatum, there were no exercise-
induced effects on D1 receptor protein expression. These results are consistent with our
previous data showing an exercise-induced increase in the expression of striatal
dopamine D2 receptor mRNA in MPTP mice (Fisher et al 2004). Taken together these
studies support that alterations in dopamine receptors, specifically D2, may underlie the
beneficial effects of exercise in PD, and suggest a mechanism by which synaptic
plasticity in the basal ganglia is facilitated through motor practice.
The purpose of studies in Chapter 4 was to combine the Golgi-Cox technique of
labeling neurons in the striatum with electron microscopy to examine the effects of high-
intensity treadmill exercise on MSN morphology, specifically dendritic spine density,
spine area and the number of synapses. Experiments used MPTP-lesioned and control
mice after 6 weeks of daily high intensity treadmill exercise. Analysis focused on MSNs
in the dorsal striatum, the region that receives rich excitatory glutamate input form the
motor cortex area involved in control of motor function. Golgi-Cox impregnation is a very
effective technique for studying both the normal and abnormal morphology of neurons
and glial cells (Graveland & DiFiglia 1985; Robinson & Kolb 1997). Using the Golgi-Cox
technique, subtle changes in dendritic architecture spine morphology can be discovered.
27
Results showed significant increases in MSN spine density in both MPTP and saline
mice exposed to treadmill exercise, this effect was more pronounced in the MPTP plus
exercise group. There was no change in the spine area measurements using electron
microscopy, however, treadmill exercise significantly increased the number of synapses
in the dorsal striatum of both treatment groups. Taken together, these results support the
hypothesis that high-intensity exercise promotes spine density increases and synapse
formation in the dorsal striatum of mice.
Studies presented in Chapter 5 focused on the morphological and physiological
correlates of the interaction between dopamine and glutamate signaling in MSNs within
the indirect pathway of the dorsal striatum in response to high intensity treadmill
exercise. The experimental approach utilized a transgenic mouse line with eGFP
expression selective to dopamine D2 receptor containing MSNs. Control mice (saline-
injected) and lesioned mice (MPTP-injected) were trained to run daily on a mouse
treadmill for 6 weeks, starting 5 days after MPTP lesion. Correlates of experience
dependent synaptic plasticity were measured by utilizing the tools of electrophysiology,
biocytin labeling, and protein expression assays. Results indicate that treadmill exercise
(a) increases spine density on MSNs within the indirect pathway of the striatum, (b)
modulates AMPA receptor subunit composition in MSNs of the indirect striatal pathway,
and (c) promotes AMPA GluR2 subunit insertion into the postsynaptic density of MSNs
in MPTP mice after treadmill exercise.
Parkinson’s disease causes pathological neuroplasticity in the basal ganglia
Dopamine originates from two groups of cells in the midbrain - substantia nigra
pars compacta (SNpc) and the ventral tegmental area (VTA). The first group of neurons
sends dopamine projections to the striatum via the nigrostriatal pathway, while the
28
second projects dopamine to the nucleus accumbens (NAc) and the cortex via the
mesocortical pathway. These two anatomically distinct pathways regulate different
behaviors. The nigrostriatal projections are primarily involved in control of motor
behavior, while the mesocortical projections modulate cognition, motivation, and reward
(Carr & White 1986; Girault & Greengard 2004).
The main loci of serotonin producing neurons in the brain are the dorsal and
medial raphe nuclei. Axonal projections of these neurons innervate the majority of brain
regions. Neurons originating in the dorsal raphe nucleus release serotonin in the cortical
and striatal regions, while neurons from the medial raphe nucleus supply serotonin to the
limbic regions (Jacobs & Azmitia 1992). Dysfunction in serotonin activity throughout the
brain leads to development of anxiety and depression in humans and animals (Mann
1999).
Parkinson’s disease (PD) is a common neurological disorder, with the average
age of onset at 55 years. It is caused by degeneration of dopamine producing neurons
within the SNpc, resulting in severe and progressive dopamine loss in the caudate
nucleus and the putamen (commonly referred to as the striatum in rodents). A critical
threshold of dopamine loss (estimated at 50-75%) causes irreversible motor symptoms,
including bradykinesia (slowness of movement), rigidity and freezing. In addition,
dopamine loss in brain regions outside of the basal ganglia leads to the development of
non-motor symptoms, ranging from depression and anxiety to aggressive behavior,
memory loss and dementia (Cummings 1992; Dauer & Przedborski 2003; Hornykiewicz
1966).
Experimental Parkinsonism was developed for research purposes by introducing
various neuronal toxins into animal models such as mice, rats and non-human primates.
Each one of these animal models has its advantages and limitations. However, all of
29
them have been well described and used to address a wide variety of research
questions. One of the commonly used neurotoxins used today in animal research is 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Accidental exposure to MPTP
caused irreversible parkinsonism in humans (Langston et al 1983; Langston et al 1999).
Mice and non human primates treated with MPTP develop hallmark pathological
features of PD, including substantial loss of dopamine producing neurons in the midbrain
and severe dopamine depletion in the striatum (Langston et al 1984; Petzinger &
Jakowec 2004). In addition, non human primates exposed to MPTP develop severe
motor complications, similar to those observed in patients with PD (Petzinger et al 2005).
However, motor symptoms in MPTP-lesioned mice are more difficult to observe and
require utilization of specific mouse motor tests in order to be examined for research
purposes.
Motor and non-motor symptoms in PD
Loss of dopamine and dopamine-producing neurons in the midbrain are hallmark
pathological signs of PD. However, other neurotransmitter systems degenerate or
become dysfunctional during the course of the disease, such as those of serotonin and
noradrenalin.
Affective behavior disorders are reported for 30 to 60% of patients with PD and
include loss of semantic and episodic memory and executive functions, depression, and
anxiety (Guze & Baxter 1986; Santamaria et al 1986; Walsh & Bennett 2001). A number
of brain regions have been implicated in development of non-motor behavioral
symptoms, including the basolateral amygdala, nucleus accumbens, frontal cortex,
dorsal and medial raphe nucleus and striatum (D'Amato et al 1987; Jellinger 1987;
Raisman et al 1986). Serotonin, projecting from the dorsal and medial raphe nuclei, is
30
thought to play a central role since perturbation in normal individuals can lead to
depression, anxiety, and memory impairment (Mayeux 1990; Pillon et al 1989b; Ressler
& Nemeroff 2000). Patients suffering from PD also develop serotonin dysfunction
demonstrated by low cortical serotonin levels and degeneration of the dorsal raphe
nucleus (Agid et al 1989; Brown & Marsden 1986; Kostic et al 1987; Scatton et al 1983).
Interactions of serotonin and dopamine throughout the basal ganglia and the limbic
system underscore their importance in mediating affective disorders in PD. Specifically,
serotonin and dopamine systems share the same biosynthetic enzymes and neuronal
circuits within the basal ganglia, midbrain, limbic system and frontal cortex and
disturbance in one can cause dysfunction in the other (Frazer & Hensler 1990; Tork
1990). Several studies have shown that 6-hydroxydopamine (6-OHDA) when
administered to early postnatal rats can lead to significant hyper-innervation of serotonin
projections to the striatum when rats are examined as adults (Berger et al 1985; Snyder
et al 1986; Stachowiak et al 1984). MPTP administration to mice and non human
primates causes significant decreases in striatal and extra-striatal serotonin levels in
addition to producing severe dopamine depletion. For example, chronic MPTP-treatment
in monkeys has been shown to cause decreased levels of serotonin in the caudate
nucleus, putamen, nucleus accumbens, hypothalamus and cortical areas (Frechilla et al
2001; Perez-Otano et al 1991; Pifl et al 1991; Russ et al 1991). In mice, acute
administration of MPTP leads to significant serotonin loss in the striatum and frontal
cortex as measured one week after lesion without cell loss in the dorsal and medial
raphe nuclei (Gupta et al 1984; Hara et al 1987; Rousselet et al 2003; Sundstrom et al
1987; Vuckovic et al 2008). However, little is known about how changes in the serotonin
system develop over time in response to dopamine loss and how they relate to
development of affective disorders in PD.
31
Results of the studies presented in Chapter 2 of this dissertation provide insight
into dynamic changes of the serotonin and dopamine systems in multiple brain regions
following MPTP lesion in the mouse. Also, the results indicate that there is a link
between development of associative memory impairment and conditioned fear with low
levels of dopamine and serotonin in the frontal cortex, ventral striatum and amygdala.
Exercise and the brain: Initiating adaptive neuroplasticity
Over the past two decades, a number of studies in humans have reported a
positive impact of exercise on brain health and function. Participation in regular exercise
programs improves cognition in older populations (Berkman et al 1993; Blomquist &
Danner 1987; Hill et al 1993). Physical activity also decreases the risk of cognitive
impairment, dementia and AD (Laurin et al 2001). Clinical studies in individuals with PD
have reported that physical exercise has positive influences on motor symptoms
(Comella et al 1994a; Miyai et al 2002; Schenkman et al 1998; Toole et al 2005).
Animal research supports the findings from human studies. Basic research
studies show that physical exercise and enriched environments have beneficial effects
on the brain and promote synaptic plasticity. Voluntary running promotes learning,
memory formation, cell proliferation and neurogenesis in the hippocampus of adult mice
and rats (Creer et al ; Farmer et al 2004; Neeper et al 1995; Pereira et al 2007; van
Praag et al 1999a; van Praag et al 1999b; van Praag et al 2005). Rearing rats in
complex environments influences cerebral cortex microvasculature, astocytic
morphology, dendritic branching patterns and synaptic number and structure
(Greenough et al 1987). Neuronal effects of complex environment rearing were not
observed uniformly across the brain, but in targeted regions such as the cerebral cortex.
32
In addition, observed changes were not a simple response to global hormonal, metabolic
and activity increases.
The effect of exercise on an injured brain had been extensively studied in animal
models of stroke. These studies formed a foundation for our understanding of the extent
of brain plasticity following injury. Based on more than 30 years of research in humans
and animals, experience-dependent recovery processes are proven to be beneficial in
restoring the brain function after acute trauma promoting brain plasticity in the injured
cerebral cortex (Dobkin et al 2004; Forrester et al 2006; Friel & Nudo 1998; Johansen-
Berg et al 2002; Jones et al 1999a; Liepert 2006; Winstein et al 1997; Wittenberg et al
2003). Animal research studies have been repeatedly investigating biological
mechanisms that mediate exercise effects on the chronically injured brain (Fisher et al
2004; Gorton et al 2010; Petzinger et al 2007; Tillerson et al 2002b; VanLeeuwen et al
2010). Exercise and behavioral enrichment can increase neuronal survival and
resistance to a brain insult (Carro et al 2001; Stummer W et al 1994), promote brain
vascularization (Black et al 1990), stimulate neurogenesis and enhance learning (van
Praag et al 1999a; van Praag et al 1999b) and positively influence cognitive function
during aging (van Praag et al 2005). Underlying mechanisms of these beneficial effects
include increased expression of brain neurotrophic factors (Farmer et al 2004; Neeper et
al 1996), increased angiogenesis (Creer et al 2010; Isaacs et al 1992; Pereira et al
2007; van Praag et al 2005), dendritic spine density and complexity (Stranahan et al
2007) and synaptic plasticity (Farmer et al 2004; van Praag et al 1999a).
Parameters of the “activity” or exercise that have emerged as being critical for
inducing neuroplasticity in the CNS with potential lasting effects are, i) intensity of
training sessions, ii) specificity for the targeted brain region, iii) difficulty of a given motor
task and iv) complexity (Fisher & Sullivan 2001; Will et al 2004). Although these
33
parameters were first established in stroke rehabilitation clinical practices and have been
proven to be important for recovery of the sensory-motor cortex, emerging studies in
patients with PD are supporting these parameters as important for functional recovery of
the injured basal ganglia.
Task specificity for an affected brain region is possibly the most important
parameter to be considered in studies of activity-dependent neuroplasticity. Different
brain regions control different behaviors. While many brain areas can be simultaneously
activated during a particular task, not all behavioral activities have the capability to
promote synaptic plasticity in all brain regions. For example, memory training can induce
synaptic plasticity in the hippocampus, the brain region degenerated in Alzheimer’s
disease (van Praag et al 2000). Likewise, training of the affected limb or body part
induces synaptic plasticity in the motor cortex following stoke (Jones et al 1999).
Therefore, behavioral activities with the most potential for inducing and maintaining
synaptic plasticity in the basal ganglia could be balance and gait training, learning
sequential motor tasks and new motor skills (Yin et al 2009), and coordinated
movements of multiple body parts (Fisher et al 2008; Petzinger et al 2010; Petzinger et
al 2007). Results of the studies presented in Chapters 3, 4 and 5 of this dissertation
provide evidence to support this hypothesis.
Intensity of an exercise task is defined as the number of repetitions and the
speed of completing the task. A high number of repetitive movements ensure high
frequency activation of motor circuits in the basal ganglia, motor cortex, and thalamus
(Swain et al 2003). Frequent neuronal activation is one of the most important factors for
lasting activity dependent synaptic plasticity. It is possible to make a parallel between
consecutive activation of brain circuits in response to repetitive behavioral activity and in
vitro electrophysiological experiments where high or low frequency electrical stimulations
34
of afferent fibers induce long lasting synaptic plasticity events in vitro, such as LTP and
LTD (Greenough 1987).
Difficulty refers to a task that is challenging and requires learning processes in
order to be completed. Learning aspects of an exercise task seem to be critical for
inducing long-term activity-dependent synaptic plasticity in targeted brain regions (Black
et al 1990).
Another parameter of exercise important for activity-dependent synaptic plasticity
is complexity. The importance of this parameter originates from observations that
enriched or complex environments induce synaptic plasticity in multiple brain regions,
such as the hippocampus, sensory-motor cortex, cerebellum and the striatum (Black et
al 1990; Briones et al 2004; Fiala et al 1978; Greenough et al 1985; Greenough &
Volkmar 1973; Jones & Greenough 1996; Kleim et al 1998; Klintsova et al 2004;
Sirevaag & Greenough 1988; Wallace et al 1995; West & Greenough 1972). For
example, rearing healthy rats in enriched environment increases density of multiple head
dendritic spines on striatal MSNs by 60% compared to rats reared in single cage
environment, determined by analyzing spine density using the Golgi-Cox method
(Comery et al 1996). The biological significance of this finding was that increased
formation of multiple headed spines on MSNs reflects the strengthening of existing
synapses (via formation of parallel synapses), or alternation of connectivity patterns (via
addition of novel synaptic inputs). When complexity is applied to an exercise task, such
as an obstacle course, during an exercise routine (like balancing and running at the
same time), then it is possible that multiple brain regions will be engaged in completing
this task and promoting activity-dependent synaptic plasticity.
35
Molecular mechanisms of activity-dependent synaptic plasticity
Most of the current knowledge about molecular mechanisms of synaptic plasticity
comes from investigations of excitatory glutamate synapses on hippocampal neurons,
where the circuitry and maps of neuronal connections are well characterized (Miyamoto
2006). Experimental introduction of long-term synaptic plasticity by high frequency
stimulation protocols and induction of LTP is a well-established technique applied to
dissociated neurons, slice cultures, and neurons in vivo (Bear & Malenka 1994). These
studies have guided our current understanding of the molecular mechanisms underlying
synaptic plasticity (Abraham & Williams 2003; Bliss & Lomo 1973; Larkman & Jack
1995; Malenka & Nicoll 1999). Also, signaling cascades involved in LTD have been
investigated in studies of synaptic plasticity in the cerebellum (Evans 2007; Ito 2002;
Linden 2001). Experimental hypotheses in this dissertation were guided by recent
discoveries of molecular mechanisms of synaptic plasticity in the hippocampus.
However, it is important to have in mind that principal neurons in the hippocampus differ
from the principal striatal neurons in many aspects, including morphological and
physiological properties, presence of inhibitory interneurons, collateral connections
between MSNs and strong dopamine modulation of striatal synapses. Hence, while
many of the findings regarding the molecular mechanisms of experience-dependent
synaptic plasticity discovered on hippocampal synapses are useful in guiding the studies
of synaptic plasticity in the basal ganglia, potential limitations of direct application of this
knowledge to striatal MSNs and their connections should be taken into consideration.
For studying the effects of physical exercise on the brain, a potentially good
model system for mechanisms of experience-dependent synaptic plasticity is the cortical
pyramidal neuron. These neurons receive excitatory glutamatergic input from the
thalamus and are part of the cortico-striatal-thalamo motor loop, which makes them
36
directly involved in control of movement together with the basal ganglia. Documented
cortical plasticity in response to motor training makes cortical pyramidal neurons well
suited for studying exercise-dependent synaptic plasticity (Davis et al 2007; Day et al
1989). Angiogenesis, neurogenesis, and axonal and dendritic sprouting are processes
that underlie synaptic plasticity of cortical maps in animal models of stroke. However,
molecular mechanisms of synaptic plasticity are not completely understood in these
neurons (Day et al 2005).
The development of advanced imaging tools has finally opened the door for long-
term studies of spine development in cortical areas of the mouse brain. Recent studies
show that learning of each new motor task and acquisition of new sensory experience is
associated with formation of a new set of synapses in motor and sensory motor regions
of the cortex (Xu et al 2009; Yang et al 2009). These studies focused on dendritic spines
of cortical pyramidal neurons. In addition to new synapse formation, learning of a new
motor task was associated with formation of new spines, by means of either stabilizing
the exiting fillopodia or formation of spines de novo. Interestingly, these studies directly
showed that learning of each new task was followed by elimination of other spines,
maintaining the total number of spines on motor cortical neurons as constant. This
dynamic process, learning and formation of a new set of spines, followed by elimination
of others, seems to be preserved throughout life. Described structural changes provide
the basis to support the hypothesis that the substrate for memory formation and learning
in the brain is synapse formation and elimination (Holtmaat & Svoboda 2009).
Another model system that is beneficial for studying central nervous system
effects of exercise or behavioral enrichment is the ventral striatum. Principal neurons in
this part of the basal ganglia are MSNs, and they receive dopamine from the midbrain
and glutamate from the cortex and thalamus. There is an abundance of literature
37
reporting molecular mechanisms of drug addiction, which is a form of dopamine
dysfunction (Beurrier & Malenka 2002; Borgland et al 2004; Dong et al 2004)(Giorgetti et
al 2001; Ishikawa et al 2009; Li et al 1997; Lu & Wolf 1999; Mangiavacchi & Wolf 2004a;
b; Nelson et al 2009; Sun et al 2008; White 1995; Wolf et al 2003). Molecular
mechanisms underlying synaptic plasticity in response to environmental enrichment in
these animal models could also support exercise-dependent plasticity in striatal regions
(Hyman et al 2006; Lex & Hauber 2008; Wolf et al 2003).
Exercise and brain plasticity in animal models of PD
Exercise is beneficial in Parkinson’s disease. Yet the molecular mechanisms
responsible for these benefits are not well characterized. Studies in animal models of
PD suggest that exercise may induce a variety of synaptic plasticity mechanisms,
including modulations of neurotransmitter signaling. A study in healthy rats reported
increases in striatal dopamine D2 receptor mRNA following running on a motorized
treadmill (Foley & Fleshner 2008a). Dopamine receptors are critical modulators of basal
ganglia circuits, yet relatively little is known about synaptic plasticity mechanisms
influencing their expression in MSNs and the role of behavioral activity in this process.
Studies presented in Chapter 3 of this dissertation focused on dopamine receptor
expression and availability for ligand binding in the dorsal striatum of MPTP mice
following 6 weeks of high intensity treadmill exercise. The results of these studies
suggest that upregulation of dopamine D2, but not D1 receptor, is a potential mechanism
of activity-dependent synaptic plasticity in dopamine-depleted striatum.
Rats with unilateral lesion of the basal ganglia by 6-hydroxydopamine (6-OHDA)
have been used extensively as animal models of PD. This neurotoxin selectively kills
dopamine producing midbrain neurons following injection directly into the brain (usually
38
only into the one hemisphere, as bilateral lesion causes high mortality due to difficulties
with eating and drinking). Rats treated with 6-OHDA develop severe motor symptoms as
cell death and dopamine depletion progress over the course of weeks. Among other
motor deficits, preferential use of the forelimb ipsilateral to the side of injury is well
documented in these animals (Tillerson et al 2002b; Tillerson et al 2001). In efforts to
investigate if physical activity is beneficial during the course of recovery from a 6-OHDA
injury, Tillerson and colleagues investigated motor behavior and brain neurochemistry in
rats forced to use the impaired forelimb. Lesioned rats were forced to use the impaired
forelimb by placing a plaster cast over the unaffected limb during the first 7 days
following brain injury. Motor behavior and dopamine levels in the striatum were
significantly improved compared to lesioned animals without casts (Tillerson et al 2001).
A follow-up study reported that early forced impaired limb use in rats with mild dopamine
depletion is neuroprotective and can attenuate dopamine cell loss when 6-OHDA is
administrated for the second time (Tillerson et al 2002b). Another type of physical
activity investigated by the same research group was motorized treadmill exercise. Two
animal models of basal ganglia injury were used in the study: 6-OHDA lesioned rat and
MPTP-lesion mouse. Mice and rats were placed on motorized treadmills two times per
day for a total of 10 days. Exercise started one day after the lesion. Neurochemical
analysis of dopamine and its metabolites in the striatum revealed attenuation of
dopamine loss in animals involved in treadmill exercise. Levels of dopaminergic terminal
markers, tyrosine hydroxylase, dopamine transporter (DAT), and vesicular monoamine
transporter 2 (VMAT2) were preserved or upregulated in exercised animals but severely
depleted in sedentary lesioned controls (Tillerson et al 2003). Elevated expression levels
of these molecular markers indicate that early treadmill exercise intervention following
39
the basal ganglia injury protects dopamine producing midbrain neurons from dysfunction
and neurodegeneration.
Relatively little is known about molecular mechanisms and cellular signaling
underlying exercise effects on the brain, and even less about the effects of exercise on
MSNs in dopamine depleted striatum and in the injured basal ganglia. Studies using the
MPTP mouse showed that high intensity treadmill exercise downregulates expression
levels of the dopamine transporter (DAT) in the striatum of both MPTP-lesioned and
control mice, and upregulated the expression of dopamine receptor D2 mRNA (Fisher et
al 2004). This observation supports the hypothesis that in the case of the dopamine
depleted basal ganglia and striatum, treadmill exercise stimulates the brain to utilize the
small amounts of dopamine left in the striatum in the most efficient way possible. Indeed,
this hypothesis has been tested by using fast scan cyclic voltometry in acute striatal
slices from adult mice after MPTP lesion, followed by 6 weeks of daily treadmill exercise.
Electrophysiological recordings showed that exercise increased local dopamine release
in the dorsal striatum without changing total dopamine tissue concentration, as
measured by HPLC (Petzinger et al 2007). A follow-up study further investigated
electrophysiological and molecular changes within striatal MSNs in response to treadmill
exercise, using the same paradigm. Whole cell patch clamp recordings showed that in
MPTP lesioned mice, exercise significantly decreased the excitability of MSNs in the
dorsal striatum in response to a stimulus from cortical afferents (VanLeeuwen et al
2010). Reduced excitability of MSNs was accompanied by increased expression of
AMPA GluR2 receptor subunits. Due to an arginine side chain residue in the pore
forming site of the AMPA receptor, the presence of GluR2 subunits effectively attenuates
Ca
2+
influx into the cell. This mechanism can significantly reduce MSN excitability by
downregulating activation of Ca
2+
sensitive kinases required for phosphorylation and
40
activation of downstream signaling molecules (Menegon et al 2002; Surmeier et al 1995;
Valjent et al 2005). Treadmill exercise also increases production of neurotrophic factors,
such as BDNF and GDNF, in the dopamine-depleted striatum. In another study, rats
lesioned with 6-OHDA were forced to run on a treadmill for 4 weeks daily, and motor
behavior was tested in the cylinder task test and amphetamine-induced rotation test
(Tajiri et al 2009). Exercise training led to improvement in motor behavior, and
preservation of tyrosine hydroxylase (TH) fibers in the striatum and TH positive neurons
in the midbrain.
Molecular mechanisms of the activity-dependent synaptic plasticity in PD
Accumulating evidence suggest that physical activity is neuroprotective and
decreases the risk of chronic brain dysfunction such as occurs in PD. Animal studies
have showed that exercise and environmental enrichment increases angiogenesis and
expression levels of BDNF, GDNF and insulin-like growth factor 1 (IGF-1) in the brain
(Carro et al 2000; Cotman & Berchtold 2002; Neeper et al 1995; Oliff et al 1998).
Upregulation of these growth factors can increase brain resistance to chronic injury. In
support of this hypothesis, a study showed that 6-OHDA lesioned rats forced to use what
would be the impaired forelimb 7 days prior to unilateral basal ganglia injury showed
significant preservation of motor function, as well as dopamine and its metabolites in the
striatum (Cohen et al 2003). Upregulation of the fibroblast growth factor 2 (FGF-2) in
both hemispheres and transient increase of BDNF and GDNF in the contralateral
hemisphere were proposed molecular support mechanisms of observed neuroprotective
effects.
A potential molecular mechanism by which exercise can modulate basal ganglia
activity is by influencing the expression and availability of dopamine receptors. This
41
hypothesis has not been tested yet and is the focus of the studies presented in Chapter
3 of this dissertation. Results from a small number of studies that have investigated
dopamine receptor expression in exercised animals support this hypothesis. In healthy
rats, habitual wheel running modulates dopamine signaling in the brain by upregulating
expression of dopamine D2 receptor mRNA in the striatum and downregulating its
expression in the midbrain (Foley & Fleshner 2008b; Foley et al 2006). Another study in
MPTP lesioned mice showed that exercise-induced upregulation of dopamine D2
receptor mRNA in the dorsal striatum was specific to lesions of the basal ganglia, as it
was not observed in control mice (Fisher et al 2004). Also, treadmill exercise increased
the half-life of dopamine in the synaptic cleft by downregulating the expression of the
dopamine transporter, the main mechanism by which dopamine is removed form the
synaptic cleft (Petzinger et al 2007). A mechanism that should be taken into the account
when interpreting receptor expression studies is the effect of exercise on dopamine
release in the striatum. Small changes in dopamine availability regulate its receptors
expression. For example, dopamine loss due to neurotoxic injury in PD patients and
animal models of PD causes transient upregulation of dopamine D2 receptor expression
in the striatum (Brown et al 2005; Deutch 2006; Kaasinen et al 2003; Kaasinen et al
2000; Laihinen et al 1991; Rinne 1991; Rinne et al 1990b). The extent of upregulation is
dependent on the animal species and the severity of neurotoxin injury (Betarbet et al
2002; Bezard et al 2000; Elsworth et al 1990; Graham 1990; Graham et al 1993; Zaja-
Milatovic et al 2005).
Dopamine depletion in PD patients and animal models of PD affects expression
levels of both dopamine D1 and D2 receptors. Striatal dopamine D2 receptor levels are
typically elevated in PD patients prior to beginning dopamine replacement therapy, as
measured by PET imaging with the dopamine receptor ligand [
11
C ] raclopride (Brooks et
42
al 1992; Rinne et al 1990a). Similar observations were found in MPTP-lesioned primates
(Bezard et al 2001; Graham 1990). In the literature, there is a general agreement that D2
receptor up-regulation is a compensatory response to severe dopamine deficiency and
reflects denervation-induced super-sensitivity of striatal dopamine receptors (Betarbet &
Greenamyre 2004; Graham 1990; Przedborski 1991). Studies utilizing animals that
spontaneously recover from a neurotoxin-induced basal ganglia injury, such as MPTP-
lesioned mice and cats, suggest that up-regulated dopamine receptor expression could
lead to the observed behavioral recovery (Frohna et al 1995). However, other studies
have reported decreased expression levels of striatal dopamine D2 receptors on the
dendritic surface of MSNs in non human primates and increased expression of
dopamine D1 receptors (Guigoni et al 2007).
Dopamine D1 and D2 receptors are the predominant forms of dopamine
receptors in the striatum and are expressed on MSNs and cholinergic interneurons, as
well as on the terminals of dopaminergic neurons originating from SNpc (Gingrich &
Caron 1993). Dopaminergic signaling through striatal dopamine receptors enables
sensorimotor coordination, initiation of movement (Alexander & Crutcher 1990; Schultz
2007; Schultz & Romo 1990), procedural and motor skill memory (Seitz et al 1990),
learning processes (McDonald & White 1994) and reward (Apicella et al 2009; Schultz
1998). Dopamine receptors also play a crucial role in the pathophysiology and treatment
of Parkinson’s disease (Albin et al 1989; Starr 1995b). The majority of dopamine
receptor agonists currently in use for treatment of PD have preferential selectivity for
dopamine D2 receptors (Hurley & Jenner 2006). It is increasingly recognized that
dopamine D2 receptor signaling cascades are altered in PD, not only as a consequence
of dopamine denervation, but also in response to drug treatments used to alleviate motor
symptoms. Therefore, the functional response of dopamine receptor stimulation
43
modification in PD is not only a consequence of altered protein expression levels, but
also due to changes in receptor coupling to intracellular second messengers. Altered
receptor sensitivity leads to imbalance between the direct and indirect pathway within
the striatum, leading to motor complications following chronic treatment with L-DOPA or
dopamine receptor agonists.
In addition to dopamine input, MSNs also receive extensive glutamatergic
projections from the motor cortex areas and the thalamus. Dopamine depletion causes
dramatic changes in striatal MSN excitability and morphology (Calabresi et al 1993; Day
et al 2006; Day et al 2008; Dehorter et al 2009; Schultz & Ungerstedt 1978) and
expression of glutamate receptors (Deutch 2006; VanLeeuwen et al 2010). Studies have
demonstrated functional interaction between dopamine D2 receptor and glutamate-
mediated synaptic plasticity in the striatum (Calabresi et al 2007; Calabresi et al 1997a;
Centonze et al 2004)(Surmeier et al 2007). Moreover, recent studies have suggested
differential vulnerability to dopamine depletion of the direct and indirect pathways of the
basal ganglia. Specifically, experimental evidence suggests that not all MSNs are
equally sensitive to dopamine loss, and that those involved in the indirect pathway and
predominantly expressing dopamine D2 receptors are more vulnerable to dopamine
depletion due to differential modulation of sodium and potassium currents by dopamine
D1 and D2 receptors, leading to differences in excitability in the presence or absence of
dopamine (Day et al 2006; Gerfen 2006; Gertler et al 2008; Shen et al 2008; Shen et al
2007; Surmeier & Kitai 1993).
Rationale for studying the striatal indirect pathway in the basal ganglia
MSNs mediate the effects of dopamine and form two major efferent pathways in
the striatum: the direct and the indirect pathway. MSNs that project to GPe express
44
predominantly D2 receptors and form the circuitry of the indirect pathway (Gerfen et al
1990; Surmeier et al 1996). The other type of MSNs project to GPi and the SNpr,
express D1 receptors, and belong to the direct pathway (Figure 1.3).
Figure 1.3: Direct and indirect pathway connections in the striatum. Signals from the
motor cortex are sent to the output nuclei of the basal ganglia (SNr) via two parallel
pathways within the striatum, direct (upper panel) and indirect pathway (lower panel).
These two pathways consist of striatal MSNs predominantly expressing two different
classes of dopamine receptors, D1 (neuron labeled in orange) and D2 (neuron labeled in
green). Abbreviations: LGP – globus pallidus external, STN – subthalamic nucleus,
MGP – globus pallidus internal, SNr – substantia nigra pars reticulata.
The currently well-accepted model of PD proposes that dopamine depletion
causes increased activity of the indirect pathway MSNs and decreased activity of the
direct pathway MSNs. This causes unbalanced output from the basal ganglia to the
thalamus and interferes with the voluntary commands from the higher motor centers,
thus producing the motor symptoms of PD (Albin et al 1989). While well established, this
model has not been directly tested in animals until recently, as it was not possible to
distinguish between the two neuronal populations using the standard electrophysiology
techniques. The indirect and direct pathway MSNs are similar in their shape, size, and
45
electrophysiological properties under physiological conditions. Recent development of
the transgenic bacterial artificial chromosome (BAC) mouse line significantly advanced
the research efforts in the field (Heintz 2001). These two mouse lines, BAC-Drd1-eGFP
and BAC-Drd2-eGFP, enabled for the first time direct analysis of the two striatal
pathways (Gong et al 2003). In these mice, expression of the green fluorescent protein
(eGFP) is under control of dopamine receptors D1 and D2 selectively. Since MSNs of
the indirect and direct pathways express high levels of dopamine D2 or D1 receptors
respectively, the presence of the transgene makes them brightly fluorescent and clearly
distinguishable from the other neurons and glia in the striatum. In Chapter 5 of this
dissertation, I used the BAC-Drd2-eGFP transgenic line to investigate the effects of high
intensity treadmill exercise on the morphology and glutamate neurotransmission of the
indirect pathway MSNs under conditions of severe dopamine loss in the striatum.
Glutamate neurotransmission in PD
Glutamate is an excitatory neurotransmitter and it is the most abundant
neurotransmitter in the brain. Within the basal ganglia, glutamate facilitates
communication between cortical and thalamic neurons and the striatal MSNs, as well as
between neurons of the STN and the SNpr (Figure 1.1). The focus of research
presented in Chapter 5 is on glutamate neurotransmission from motor cortex areas to
the striatal MSNs. Glutamate acts on neurons through two types of receptors -
metabotropic and ionotropic. While metabotropic receptors are responsible for long-term
glutamate signaling on neurons, ionotropic receptors act as fast mediators of
neurotransmission and are critical for synaptic plasticity as they simultaneously act as
ion channels. There are two classes of ionotropic glutamate receptors in the brain -
NMDA and AMPA. AMPA receptors respond rapidly to glutamate in the synapse and
46
facilitate initial depolarization of the cell. At their normal resting potential (-80mV), NMDA
receptors are inactive, due to a Mg
2+
ion block inside of the ion channel pore. As a result
of the action of AMPA receptors and initial depolarization, the Mg
2+
block is released
from the NMDA receptors and they become permeable for Na
+
as well as Ca
2+
ions,
further depolarizing the neuron and allowing an action potential to occur. AMPA
receptors have been the focus of the intense research efforts in the past decade due to
their critical involvement in LTP in the hippocampus, the most extensively studied site of
activity-dependent neuroplasticity (Carroll et al 1999; Ju et al 2004; Kauer & Malenka
2006; Malenka 1994; 2003; Malinow & Malenka 2002). More recent research is focusing
on the role of AMPARs in LTD in the cerebellum and striatum (Huang et al 2008; Nicola
et al 2000; Thomas et al 2001; Thomas et al 2000).
Functional AMPA receptors are tetrameric channels composed of four different
subunits (GluR1-GluR4, or by the new nomenclature GluA1-GluA4) (Dingledine et al
1999; Washburn et al 1997). In addition, when expressed at the surface of the plasma
membrane, AMPA receptor tetramers require the auxiliary subunits called
transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon homologues
(CNIH-2 and CNIH-3) for regulated gating, ion permeability, pharmacology, and
trafficking (Brown et al 2007; Famous et al 2008; Hirling 2009; Ju et al 2004; Man et al
2000; Petrini et al 2009). Heterotetrameric receptors are predominantly found on the
surface of the neuron as dimers of dimers, such that a tetramer can be formed of two
subunits of GluR2 and the two subunits of either GluR1, GluR3 or GluR4 (Greger et al
2003; Greger et al 2007; Isaac et al 2007; Mansour et al 2001). While the four subunits
share 70% sequence identity, an important structural difference between them is the
presence of an arginine side chain in the pore forming site in the GluR2 subunit, which is
replaced by glutamine in the GluR1, GluR3 and GluR4 subunits. This single amino acid
47
side chain, the result of post-translational modification, gives a profound functional
difference to the AMPA receptors containing the GluR2 subunits. Positive charge from
arginine side chain blocks Ca
2+
entry through the channel pore, thus allowing
permeability only to Na
+
and K
+
ions. Unlike GluR2 lacking AMPA receptors that stay
blocked by intracellular polyamines at positive membrane potentials, GluR2 containing
receptors stay open and are insensitive to the polyamine block.
Trafficking and activity of AMPA receptors is crucial for spine maintenance and
activity-dependent synaptic plasticity. While the molecular mechanisms of these
processes are not well characterized, some key signaling molecules have recently been
identified. The crucial link between synaptic activity and actin phosphorylation, which
regulates spine morphology, is the family of small GTPasas, such as Cdc42, Rac-1 and
RhoA, the latter inhibiting spine growth. For their function, cycling between the active
GTP-bound form and the inactive GDP-bound form, small GTPases are dependent on
guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs).
GEFs and GAPs are major branching points of cellular signaling in dendritic spines and
their activity is coupled by protein kinases. Also, GEFs and GAPs are intermediate
signaling molecules, bridging the signaling pathways triggered by neuronal activity and
downstream pathways that regulate the actin cytoskeleton and involve two specific
protein kinases, PAK1 and PAK3. A recently identified signaling pathway that mediates
AMPA receptor trafficking with respect to spine morphology makes use of GEF-H1,
which interacts with AMPA receptors and is involved with RhoA GTPase (Kang et al
2009). The exact pathway is not yet well understood, but it seems to involve the
inactivation and endocytosis of AMPA receptors at the synapse, Ca
2+
mediated
translocation of GEF-H1 into the spine and activation of RhoA coupled with the inhibition
of Rac1. This signaling pathway leads to inhibition of spine growth at the developing
48
synapses (Li et al 2002; Ryan et al 2005). In addition, other small GTPases are involved
in AMPA receptor trafficking at the synapse. Insertion of GluR2 subunits into the AMPA
receptor tetramers at the membrane is dependent upon low levels of activity of Ras,
which is the downstream signaling molecule for the MAPK-ERK pathway. Alternatively,
high levels of Ras signaling lead to the insertion of GluR1 into the AMPA receptors at the
membrane via the PI3-kinase/PKB-Akt pathway (Qin et al 2005).
AMPA receptor trafficking and synaptic plasticity
Phosphatidylinositol-(3,4,5)-trisphosphate (PIP
3
) signaling is necessary for
functional targeting of AMPA receptors to the synapse. Prolonged downregulation of
PIP
3
impairs accumulation of PSD-95 protein to the spines and causes AMPA receptors
to mobilize from the postsynaptic density to the perisynaptic membrane within the spines
of rat hippocampal neurons. This kind of mobilization of AMPA receptors leads to
synaptic depression (Arendt et al 2010).
Throughout the brain, AMPA receptors serve as mediators of fast glutamate
excitatory neurotransmission, responding quickly to the presence of glutamate in the
synapse. Recent findings have proposed that AMPA receptors play a critical role in the
synaptic plasticity. AMPA receptors localized on membrane surface consist most heavily
of GluR1 and GluR2 subunits. Changes in the number of AMPA receptors on the
membrane modulate synaptic strength. AMPA receptor trafficking from internal
synaptosomal compartments to synaptic sites is required for synaptic plasticity. In
addition, increasing evidence suggests that changes in composition of AMPA receptors
expressed at the membrane are important mechanisms of synaptic plasticity (Conrad et
al 2008; Liu & Cull-Candy 2000; Malinow 2003; Shi et al 1999). Mechanisms of synaptic
plasticity involving the regulation of the receptor number and activity on the postsynaptic
49
side have became the focus of intense research efforts over the course of the past
decade. This mechanism of activity dependent synaptic plasticity is investigated in this
dissertation in studies presented in Chapter 5.
Summary
Taking into consideration the complexity of neuroplasticity mechanisms in the
basal ganglia triggered by dopamine depletion and activity-dependent synaptic plasticity
mechanisms that have the potential to modulate basal ganglia activity in conditions such
as PD, it is of great importance to further study these processes in animal models in
order to develop new therapies and treatments for patients affected by PD. Studies
presented in this dissertation are part of current research efforts to better understand
synaptic plasticity in PD and the cellular and molecular processes underlying activity-
dependent plasticity in the basal ganglia of the MPTP mouse model.
50
CHAPTER 2:
MEMORY, MOOD, DOPAMINE AND SEROTONIN IN MPTP-LESIONED MOUSE
MODEL OF PARKINSON’S DISEASE
1
2.1. Abstract
The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse
serves as a model of basal ganglia injury and Parkinson’s disease. The present study
investigated the effects of MPTP-induced lesioning on associative memory, conditioned
fear and affective behavior. Male C57BL/6 mice were administered saline or MPTP and
separate groups were evaluated at either 7 or 30 days post-lesioning. In the social
transmission of food preference test, mice showed a significant decrease in preference
for familiar food 30 days post-MPTP compared to controls. Mice at 7 and 30 days post-
MPTP-lesioning had increased fear extinction compared to controls. Dopamine and
serotonin were depleted in the striatum, frontal cortex, and amygdala. No changes in
anxiety or depression were detected by the tail suspension, sucrose preference, light-
dark preference, or hole-board tests. In conclusion, acute MPTP-lesioning regimen in
mice causes impairments in associative memory and conditioned fear, no mood
changes, and depletion of dopamine and serotonin throughout the brain.
2.2. Introduction
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by
motor impairment including slowness of movement, rigidity, balance dysfunction, and
resting tremor. However, disabling non-motor symptoms are seen in 30 to 60% of
patients and include semantic and episodic memory loss, impairment of executive
1
Results of this chapter have been published in Neurobiology of Disease journal (Vuckovic et al 2008).
51
function, depression, and anxiety (Cummings 1992; Hornykiewicz 1963; Pillon et al
1989a; Walsh & Bennett 2001). A number of brain regions have been implicated in
influencing non-motor behavioral symptoms, including the basolateral amygdala,
nucleus accumbens, frontal cortex, and the raphe nucleus (Ressler & Nemeroff 2000)
(Walsh & Bennett 2001). Together with dopamine, serotonin from the dorsal and medial
raphe nuclei is thought to play a central role in regulating affective behavior. Perturbation
of serotonin neurotransmission in normal individuals can lead to depression, anxiety,
and memory impairment (Mann & Yates 1986; Mann 1999; Pillon et al 1989b). Patients
with PD develop central serotonergic dysfunction, such as low cortical serotonin levels
and degeneration of the dorsal raphe nucleus (Agid et al 1989; Cummings 1992;
Gotham et al 1986; McCance-Katz et al 1992; Scatton et al 1983). 1-methyl-4-phenyl-
1,2,3,6,-tetrahydropyridine (MPTP) neurotoxicity in substantia nigra pars compacta
(SNpc) produces severe dopamine depletion in mice and non-human primates, and
causes significant decrease of serotonin across multiple brain regions. For example,
chronic MPTP-treatment in monkeys decreases levels of serotonin in the caudate
nucleus, putamen, nucleus accumbens, hypothalamus, and cortical areas (Frechilla et al
2001; Perez-Otano et al 1991b; Pifl et al 1991; Russ et al 1991). In mice, acute
administration of MPTP leads to serotonin loss in the striatum and frontal cortex one
week after lesioning (Rousselet et al 2003).
The purpose of the current study was to evaluate the effects of MPTP-lesioning
on associative memory, conditioned fear, depression and anxiety, since this neurotoxic
injury leads to depletion of dopamine and serotonin in brain regions important for these
behaviors. After acute MPTP-lesioning, mice were tested at 7 days (greatest dopamine
depletion) and 30 days (partial recovery of striatal dopamine). We used established
mouse tests for associative memory (social transmission of food preference), fear
52
conditioning, anxiety (light-dark preference, hole board), and depression (tail
suspension, sucrose preference). Brain regions involved in control of affective behavior
(frontal cortex, amygdala, and the raphe nucleus), as well as the basal ganglia (ventral
mesencephalon, striatum) were examined for levels of dopamine, serotonin and their
metabolites. We observed impairment in associative memory in mice at 30 days post-
MPTP-lesioning and increased fear extinction at both 7 and 30 days post-MPTP. Despite
significant depletion of both dopamine and serotonin at these time points, there was no
significant increase in depression and anxiety compared to control mice. Overall, these
results indicate that the acute MPTP-lesioned mouse model manifests some but not all
non-motor behaviors seen in patients with PD.
2.3. Materials and methods
Animals, treatment groups and MPTP administration
Male C57BL/6 mice 8 to 10 weeks old (Charles River Laboratories, Wilmington,
MA) and weighing between 25 and 30g were group-housed in a temperature-controlled
room under a 12h light/12h dark cycle with free access to water and standard rodent
food. All procedures were performed in accordance with the NIH Guide for the Care and
Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use
Committee at the University of Southern California. A total of 42 mice were used in this
study. For lesioning, mice received 4 i.p. injections of 20 mg/kg MPTP (free-base;
Sigma-Aldrich, St. Louis, MO) in saline at 2h intervals or 4 injections of 0.1 ml 0.9 %
NaCl as control. Mice were tested either at (i) 7 days post-saline (n=20), (ii) 7 days post-
MPTP (n=10) or (iii) 30 days post-MPTP (n=12). The degree of lesioning was
determined at the end of the study by unbiased stereological counting of the remaining
dopaminergic neurons in the SNpc. All lesioned mice had between 50 % and 75 % of
53
dopaminergic cell loss. These results are in agreement with previous reposts using the
same MPTP-lesioning regimen (Jakowec et al 2001; Petzinger et al 2001; Petzinger et al
2007; Przedborski et al 2001).
Behavioral testing
After MPTP-lesioning, mice were subjected to a series of behavioral tests for
anxiety (light-dark exploration, hole-board), depression (sucrose preference, tail
suspension), associative memory (social transmission of food preference [STFP]), and
conditioned fear. Tests were conducted over 6 days with the following order: (1) STFP,
(2) light-dark exploration, (3) sucrose preference, (4) hole-board, (5) conditioned odor
aversion, (6) tail suspension, and (7) conditioned fear (Fig 2.1). Tests which are known
to induce acute stress response (tail suspension and fear conditioning) (Brown et al
1984; Liu et al 2003; Pugh et al 1997) were conducted at the end. The order of tests was
not randomized. Any potential carry-over effects were equal between the groups as all
mice were tested in the same order. Tests occurring on the same day were conducted at
least 3h apart. Each behavioral test was administered once to each mouse. All tests
were performed in a darkened room with dim red lights and animals were allowed to
habituate to the testing room for 1h prior to each test. Details for each test are presented
in the following sections.
54
Figure. 2.1: Diagram representing the timeline and order of behavioral tests following
acute MPTP-lesioning (4 x 20 mg/kg, 2h apart) or saline injections (4 x 0.1 ml, 2h apart).
The order of behavioral tests was determined according to increased stress. Separate
groups of mice were tested at 7 or 30 days post-MPTP-lesioning.
Social transmission of food preferences (STFP) for olfactory memory was
conducted as described previously (Holmes et al 2002; Kogan et al 1997; Wrenn et al
2003). Briefly, a demonstrator mouse was randomly chosen from each home cage prior
to MPTP administration. Initially, all mice were habituated for 18h to powdered chow
presented in two 125g food jar assemblies (Dyets, Inc., Bethlehem, PA) in the opposite
corners of the home cage. During this time, standard food pellets were unavailable. One
day later, demonstrator mice were removed from their home cages, individually housed,
and food-deprived overnight with free access to water. The next day, each demonstrator
mouse received powdered chow mixed with either 1% cinnamon or 2% cocoa (w/w) for
1h, or until at least 0.2g of powdered food was consumed. To avoid a bias in the cued
flavor, half of the demonstrators randomly received cinnamon- and the other half cocoa-
flavored food. Immediately afterwards, demonstrator mice were returned to their home
cages to interact with observer mice for 30min. At the end of the interaction period,
demonstrator mice were removed. Testing of food preference in observer mice took
place 24h later, following overnight food deprivation with free access to water. During the
test, observer mice were caged individually and were given a free choice of food
55
flavored with 1% cinnamon or 2% cocoa. To control for possible place preference, the
position of the food jar assemblies with the cued flavor was balanced between cages.
Observer mice were allowed to eat for 1h and food consumption from each jar was
determined by weight. The percent of total food intake consumed as the cued flavor was
determined.
The light-dark exploration test for anxiety was conducted as previously
described (Holmes et al 2002). The test uses the ethological conflict between the
tendencies of mice to explore a novel environment and to avoid a brightly lit open area.
This test has been shown to be sensitive to changes in serotonergic tone (Holmes et al
2002). A standard polypropylene mouse cage (30 x 19 x 13 cm) was divided with an
opaque partition containing a small opening at the bottom (8 x 5 cm) into a larger light
chamber and a smaller dark chamber. The light chamber (20 x 19 x 13 cm) was
transparent and brightly illuminated by a 60-watt bulb placed 40 cm above the cage top.
The dark chamber (10 x 19 x 13 cm) was black and closed at the top with a black
Plexiglas lid. The test was conducted in a soundproof room and the apparatus was
cleaned with warm water and 70% ethanol between each mouse. Each mouse was
placed in the lighted chamber facing away from the entrance to the dark chamber, and
its behavior was recorded on video for 5 min. Measurements were obtained for: (i)
latency to first enter the dark chamber, (ii) time spent in the dark, and (iii) number of
transitions between the two compartments. A transition was considered only when a
mouse entered into a compartment with 3 or more paws.
The sucrose preference test for depression was performed as a modification of
the 2-bottle preference test for mice (Strekalova et al 2004). Mice were deprived of food
and water overnight and placed in separate cages 1h before the start of testing. Mice
were offered solutions of 1% sucrose (Sigma-Aldrich, MO) or tap water for 1h. Fluid
56
consumption was determined by weight and expressed as percent of total fluid intake
consumed from the sucrose solution. The positions of the water and sucrose bottles
were alternated to control for side preferences.
The hole-board test for anxiety based on exploratory behavior was performed
as previously described (Boissier & Simon 1962; do-Rego et al 2006). This test is
frequently used as an indicator of directed exploratory behavior in rodents (Crawley
1985). The testing apparatus consisted of a 2cm-thick square plastic board, 40 × 40cm,
with 16 holes (2cm diameter) regularly spaced 7cm apart over the surface and 3.5cm
from the edges (Ugo Basile, Italy). The board was positioned 50cm above floor level.
Each animal was placed at the corner of the board and allowed to freely explore for 5
min. The number and location of head dips was recorded using a video camera and a
trained observer blinded to the treatment group scored videotapes. A head dip was
considered when a mouse placed its head into a whole up to the neck. Between testing
of each mouse, the board surface was cleaned with water and 70% ethanol.
The conditioned odor aversion with isoamyl-acetate was used as a rapid
assessment of odor detection in mice. The one bottle test was performed as previously
described (Passe & Walker 1985; Wright & Harding 1982) with minor modifications. Mice
were deprived of food and water overnight and housed separately 1h before the start of
testing. Testing consisted of four 10min trials. Breaks between the trials lasted 30 min.
During the first two trials, mice were allowed to drink water containing both 0.1% (v/v)
isoamyl acetate (artificial banana aroma) and 0.5% (w/v) quinine hydrochloride (bitter
taste) (Sigma-Aldrich, MO). During the third trial, mice were tested for avoidance of
isomayl acetate odorized water without quinine. The last trial consisted of tap water.
Fluid consumption was determined by weight. Preference ratio for isoamyl acetate was
determined from the last two trials as follows: odorized water (g) / odorized water + tap
57
water (g). Preference ratio of below 0.5 indicates aversion, and therefore detection of the
odorant.
The tail suspension test for depression was preformed as previously described
(Steru et al 1985). This test relies on immobility as a measure of “behavioral despair”
once the mouse perceives that the escape from the apparatus is impossible. Mice were
individually suspended by their tails at a height of 20cm using a piece of adhesive tape
wrapped around the tail, 2cm from the tip. Behavior was videotaped for 6 min. The
duration of immobility was measured using a stopwatch. Mice were considered immobile
only when hanging completely motionless. Mice that climbed up their tails were
excluded. Results were expressed as percent of time spent immobile.
Auditory conditioned fear response was assessed as previously described
(LeDoux 2000). The test consisted of an 8 min acquisition phase on the first day and an
8 min extinction phase on the next day. Training was conducted in a soundproof room
with dim red light and background noise level of 50dB. Each mouse was placed in the
middle of a testing chamber (23cm × 20cm × 20cm) with an electrified metal rod floor
(2mm diameter, 6mm separation). The chamber was cleaned with water and 70%
ethanol before testing each mouse. Fear acquisition consisted of a 3 min acclimation
period, 3 pairings of tone/foot shock separated with 1 min quiet intervals, and 1 min quiet
consolidation period at the end of testing. For each pairing of tone/foot shock, mice were
presented tone (30s of 80dB, 1000Hz/8000Hz continuous alternating sequence of
250ms pulses) generated using LabView 7.1 software (National Instruments
Corporation, Austin, TX) and delivered through speakers on the top of the testing
chamber. Each tone was immediately followed by a mild foot shock (2s, 0.6mA).
Freezing behavior (no visible movement except for respiration) was recorded on
videotape during the test. The extinction phase was conducted the next day in a different
58
room illuminated with blue indirect light. Each mouse was placed in a cylindrical
Plexiglas observation chamber (diameter 28cm) with a smooth Plexiglas floor. Following
an initial 2 min acclimation period, recall and extinction of freezing in response to the
tone (presented continuously for 6 min) was monitored in the absence of foot shock. The
duration of the freezing response in seconds was measured within each 1-minute
interval of both acquisition and extinction phases. Freezing behavior was manually
scored using Observer XT version 6.1.35 software (Noldus Information Technology, San
Diego, CA).
Tissue preparation
Brains were collected 24h after the last behavior test. For immunohistochemistry,
a subset of mice (n = 4 per group), were sacrificed by pentobarbital overdose and
transcardially perfused with 4% paraformaldehyde, post-fixed in the perfusion fixative for
48h at 4°C, cryoprotected in 20% sucrose for 24h, frozen in isopentane on dry ice, and
stored at -80°C. For HPLC analysis, another subset of mice (n=5 per group) was killed
by cervical dislocation. Brains were quickly removed and regions of interest identified
using a standard mouse brain atlas (Paxinos 2001). Frontal cortex (rostral to Bregma
2.50), dorsal and ventral regions of mid-striatum (including the nucleus accumbens),
amygdala, ventral mesencephalon (containing SNpc, SNpr and VTA) and the raphe
nucleus (dorsal and medial part) were rapidly dissected, immediately frozen in
isopentane on dry ice and stored at -80°C.
Neurochemical analysis
Neurotransmitter concentrations of dopamine, 3, 4-dihydroxyphenylacetic acid
(DOPAC), homovanilic acid (HVA), serotonin, and 5-hydroxyindoleacetic acid (5-HIAA)
59
were determined by HPLC with electrochemical detection as previously described (Irwin
et al 1992; Kilpatrick et al 1986; Petzinger et al 2007). The system consisted of an ESA
auto-sampler (ESA Inc., Chelmsford, MA) equipped with a 150 x 3.2 mm reverse phase
C-18 column (3µm diameter) regulated at 28°C and a CoulArray 5600A (ESA Inc,
Chelmsford, MA), equipped with a 4-channel analytical cell with potentials set at -100
mV, -30 mV, 220 mV and 350 mV. The HPLC was integrated with a DellGX-280
computer with CoulArray analytical program for Windows (ESA Inc, Chelmsford, MA).
Mobile phase consisted of acetonitrile in phosphate buffer and an ion-pairing agent and
was delivered at 0.6ml/min rate. Fresh frozen tissue was homogenized in 0.4 M HClO
4
,
and centrifuged to separate precipitated protein. The pellet was resuspended in 0.5 M
NaOH and used to determine total protein concentration with the CoomassiePlus protein
assay (Pierce, Rockford, IL) and microplate reader ELx800 (BioTek Instruments Inc.,
Winooski, VT) equipped with KCjunior software.
Immunohistochemical staining
Analysis of relative expression of striatal tyrosine hydroxylase (TH)
immunoreactivity was carried out as previously described (Jakowec et al 2004a;
Petzinger et al 2006; Petzinger et al 2007). Briefly, coronal brain sections were cut at
25µm thickness through the mid-striatum and collected in phosphate-buffered saline
(PBS, pH 7.2). Sections were exposed to rabbit polyclonal anti-tyrosine hydroxylase
antibody (1:5000, Chemicon, Temecula, CA) for 24h at 4°C followed by 2h incubation in
IRDye700 conjugated goat anti-rabbit IgG (1:2500, Molecular Probes, Eugene, OR).
Following extensive washing, sections were mounted on gelatin-coated slides and
scanned using LI-COR Odyssey near infrared imaging platform (LI-COR Biotechnology,
Lincoln, NE). Multiple brain sections at the mid-striatum (4 to 5 sections per mouse) from
60
4 mice per group were prepared and analyzed in parallel. Fluorescence intensity within
an oval shaped region of interest (1mm
2
) in dorsal striatum was measured and corrected
for background by subtracting the adjacent corpus callosum. Values for treatment
groups were normalized to saline animals prior to statistical analysis.
Unbiased stereological counting of SNpc dopaminergic neurons
The degree of MPTP-lesioning at 7 and 30 days post-MPTP lesioning was
determined by stereological counting of dopaminergic neurons in SNpc. For this
purpose, coronal sections were collected
started rostral to the substantia nigra at bregma
–2.50
mm before the closure of the third ventricle through to the
prominence of the
pontine nuclei at bregma –4.24 mm according
to the stereotaxic atlas of the mouse brain
(Paxinos 2001). Every sixth section from 4 mice per group was included in the analysis.
Sections were exposed to rabbit polyclonal anti-tyrosine hydroxylase antibody (1:5000,
Chemicon, Temecula, CA) for 24h at 4°C followed by and avidin-biotin complex (ABC
elite Kit, Vector Labs, Burlingame, CA). Staining was visualized by exposure to 3,
3’diaminobenzidine tetrahydrochloride (Pierce, Rockford, IL), after which sections were
mounted on gelatin-coated slides, and cover-slipped. Cell nuclei were visualized by
cresyl-violet staining. The SNpc was delineated
from the rest of the brain based on TH-ir.
Sections were examined using Olympus BX-50 microscope (Olympus
Optical, Tokyo,
Japan) equipped with a motorized stage and digital
Retiga-cooled CCD camera (Q-
Imaging, Burnaby, British Columbia,
Canada). Each stained ventral mesencephalon
section was viewed
at low magnification (4x) and the SNpc outlined and
delineated from
the ventral tegmental-immunoreactive neurons
using the third nerve and cerebral
peduncle as landmarks. Neurons
were viewed at higher magnification (40x) and counted
if they displayed TH-ir and had a clearly defined nucleus, cytoplasm,
and nucleolus.
61
Analysis was performed
with the computer-imaging program BioQuant Nova Prime
(BioQuant
Imaging, Nashville, TN). The total number of SNpc dopaminergic neurons
was
determined based on the method of Gundersen and Jensen (1987).
Statistical analysis
With the exception of fear-conditioning, all results were evaluated by a one-way
analysis of variance (ANOVA) and Bonferroni multiple comparison test when
appropriate, or by one-way ANOVA on ranks followed by Dunn’s post-hoc test when the
normality test or equal variances test failed. Software used for statistical analysis was
Prism5 for Windows (Graph Pad Software Inc., San Diego, CA). A repeated measures
ANOVA was used to analyze freezing behavior from the fear-conditioning test. Data from
all experiments are presented as mean ± SEM and p<0.05 was considered significant.
2.4. Results
Social transmission of food preference
Associative olfactory memory was assessed using the STFP test (Fig. 2.2).
When presented with 2 unfamiliar flavors of powdered food, control mice strongly
preferred the flavor previously consumed by the demonstrator mouse (79.0 ± 3.7% of
total food intake). Lesioned mice tested 7 days after MPTP showed a similar preference
(79.0 ± 3.3%). However, preference for the demonstrated flavor declined significantly in
mice tested 30 days post-MPTP (58.7 ± 6.3%) (F
(2,41)
=5.614; p<0.05). As the STFP test
relays on the ability of mice to discriminate odors, it was important to test if all mice had
similar olfactory function. For this purpose, the conditioned odor aversion test with
isoamyl acetate was used. Mice from all three groups avoided water odorized with
isoamyl acetate (preference ratio for saline: 0.4 ± 0.1; 7 days post-MPTP: 0.2 ± 0.1; 30
62
days post-MPTP: 0.3 ± 0.1). Preference ratio below 0.5 indicates the ability to
discriminate odors. Based on these results, mice in this study had no impairment in
olfactory function.
Fig. 2.2: Associative memory impairment in MPTP-lesioned mice measured by the
social transmission of food preference test. The preference for familiar food was tested
in control (n=20) and lesioned mice at 7 days (n=10) and 30 days post-MPTP (n=12).
Data are presented as mean ± SEM of percent preference for familiar food. The symbols
“*” and “
#”
represent statistically significant difference compared to the control and 7 days
post-MPTP group, respectively (p<0.05).
Light-dark preference and hole-board
Light-dark preference and the hole-board tests were used to determine levels of
anxiety in mice 7 and 30 days post-MPTP-lesioning (Fig. 2.3). Mice from all three groups
showed a similar preference for the dark compartment (F
(2,39)
= 1.428; p>0.05) during the
light-dark exploration test (Figure 2.3A). Saline-treated mice spent 66.1 ± 3.7% of the
time in the dark. Likewise, mice at 7 days post-MPTP-lesioning spent 74.5 ± 3.6% of
time in the dark and those tested at 30 days post-MPTP-lesioning spent 65.4 ± 3.3% of
the time in the dark. The average number of transitions between the light and dark
compartments was similar in all three groups (13.1 ± 1.6 for saline mice; 12.6 ± 1.3 and
16.0 ± 1.9 for mice 7 days and 30 days post-MPTP-lesioning, respectively; F
(2,39)
=
63
1.246; p>0.05). There was no significant difference between the latency to first enter the
dark compartment between groups (26.7 ± 5.3 s for saline mice; 15.9 ± 4.2 s and 25.6 ±
5.6 s for mice 7 days and 30 days post-MPTP-lesioning, respectively; F
(2,39)
= 0.9846;
p>0.05). Exploratory behavior was measured in the hole-board test as a second test of
anxiety (Fig. 2.3B). As measured by head dipping, saline treated mice visited 24.8 ± 2.2
holes during the 5 min test, similar to mice at 7 days post-MPTP-lesioning (24.5 ± 3.0
holes). Mice tested at 30 days post-MPTP-lesioning had 33.0 ± 3.1 head dips. The
difference in the total number of head dips between groups was not statistically
significant (F
(2,33)
= 3.053; p>0.05).
Figure 2.3: Anxiety in mice 7 and 30 days post-MPTP-lesioning measured in light-dark
preference and the hole-board tests. Data are presented as mean ± SEM from control
(n=20), 7 days (n=10) and 30 days post-MPTP (n=12). (A) Time spent in the dark
compartment during the 5 min light-dark preference test (in % or total time). (B) The total
number of head dips during 5min hole-board test.
Sucrose preference and tail suspension
Sucrose preference and the tail suspension tests were used to determine levels
of depression in mice after MPTP-lesioning (Fig. 2.4). In particular, the sucrose
preference test measures anhedonia following overnight water deprivation (Strekalova et
al 2004). Mice from all three groups had high preference for a 1% sucrose solution (80-
64
85%) compared to the tap water (15-20%) (Fig. 2.4A). There was no significant
difference in the amount of sucrose consumed (F
(2,38)
= 0.968; p>0.05). Furthermore, all
mice had similar fluid intake during the 1h testing period (saline mice: 0.9 ± 0.1g; 7 days
post-MPTP-lesioning: 0.8 ± 0.1g; and 30 days post-MPTP-lesioning: 1.1 ± 0.1g;
F
(2,37)
=1.268; p>0.05). The tail suspension test measures behavioral despair (Steru et al
1985). Saline-treated mice spent 36.6 ± 3.1% of time in passive immobility (Fig. 2.4B).
Similarly, mice at 7 days post-MPTP-lesioning spent 29.3 ± 3.3% of time in immobility.
Interestingly, mice tested at 30 days post-MPTP-lesioning spent significantly more time
immobile (44.3 ± 3.2%) compared to the 7 days post-MPTP group (F
(2,39)
= 4.372;
p<0.05), however, this was not statistically different compared to saline-treated mice.
Only one out of 19 mice tested in the control group was excluded from the test because
it climbed up its tail. None of the MPTP-lesioned mice were excluded from the test.
Figure. 2.4: The effect of acute MPTP-lesioning on depression in mice 7 and 30 days
post-treatment measured using sucrose preference and tail suspension tests. Data are
presented as mean ± SEM of (A) preference for 1% sucrose and (B) time spent in
immobility (in % of total time) for control (n=18), 7 days (n=10) and 30 days post-MPTP
(n=12). The symbol “
#”
represents statistically significant difference compared to 7 days
post-MPTP group (p<0.05).
65
Conditioned fear
Acquisition and extinction of conditioned fear response was measured using the
auditory fear conditioning test. All mice showed little or no freezing during the baseline
period of acquisition session (percent time freezing for saline: 1.4 ± 0.4%; for 7 days
post-MPTP: 0.2 ± 0.1%; for 30 days post-MPTP: 0.7 ± 0.4%). During subsequent
pairings of tone and foot shock, all mice showed increase of the freezing behavior. Mice
in all three groups had a similar levels of freezing after the third foot shock (saline: 54.9 ±
10.7%; 7 days post-MPTP: 46.2 ± 10.5%; 30 days post-MPTP: 49.4 ± 4.1%; F
(2,16)
=
0.249; p>0.05). The next day, (Fig. 2.5), the baseline freezing response of all mice was
similar (percent freezing for saline: 23.7 ± 6.2%; 7 days post-MPTP: 9.6 ± 2.4%; 30 days
post-MPTP: 18.5 ± 8.3%). At the onset of auditory stimulus (without foot shock), all mice
showed a robust increase in the freezing response (saline: 68.8 ± 3.1%; 7 days post-
MPTP: 56.8 ± 6.7%; 30 days post-MPTP: 51.0 ± 6.2%). However, after 6 minutes of
continuous tone exposure, both groups of MPTP-lesioned mice spent significantly less
time freezing (7 days post-MPTP: 9.5 ± 3.2%; and 30 days post-MPTP: 17.5 ± 8.2%)
compared to saline controls (43.7 ± 6.0%). Repeated measures ANOVA followed by
Bonferroni post-test showed significant difference in freezing response over time
between saline-treated and MPTP-lesioned mice at both 7 and 30 days (F
(2,17)
= 23.08;
p<0.05).
66
Fig. 2.5: Acquisition and extinction of fear response in MPTP-lesioned mice measured in
the fear conditioning test. Fear-induced immobility was measured in control (n=5), 7
days (n=6) and 30 days post-MPTP mice (n=8). Data are presented as mean ± SEM of
the freezing response (% of 2 or 4 min periods). Following 2min baseline, continuous
tone was played for 6 min without foot shock (tone onset: min 3-4; tone continuation:
min: 5-8). The symbol “
*”
indicates statistically significant difference compared to control
group (p<0.05).
HPLC analysis of dopamine, serotonin, and their metabolites
HPLC analysis was used in tissue homogenates to determine the levels of
dopamine and its metabolites (DOPAC and HVA), as well as serotonin and its metabolite
5-HIAA. The turnover ratio for dopamine was determined as ([DOPAC] + [HVA]) /
[dopamine] and for serotonin as [5-HIAA] / [serotonin]. Six brain regions were analyzed
including the frontal cortex, dorsal striatum, ventral striatum, ventral mesencephalon
(VME), amygdala, and the raphe nucleus (Table 2.1 and Figure 2.6). Taken together,
MPTP-lesioned mice had severe dopamine depletion in the dorsal and ventral striatum
and frontal cortex, and no significant loss in the amygdala, VME or the raphe nucleus.
The dopamine greatest depletion was measured at 7 days post-MPTP. Serotonin was
significantly depleted in the dorsal and ventral striatum, frontal cortex, and amygdala.
The greatest loss of serotonin was measured at 30 days post-MPTP-lesioning. There
67
was no significant change in the level of the serotonin metabolite 5-HIAA in any of the
examined brain regions.
Dopamine and serotonin levels measured by HPLC and their calculated turnover ratios in six brain regions
of the MPTP-lesioned and control mice.
Treatment DA DA Turnover 5-HT 5-HT Turnover
Dorsal Striatum Control 141.3 + 12.4 0.2 + 0.0 7.1 + 0.8 1.0 + 0.1
7 d post-MPTP 7.5 + 1.6* 1.1 + 0.5 3.7 + 0.5* 2.4 + 1.0
30 d post-MPTP 19.9 + 5.2* 1.4 + 0.3 3.3 + 0.4* 2.9 + 0.5
Ventral Striatum Control 91.1 + 7.7 0.2 + 0.0 16.4 + 1.8 0.7 + 0.1
7 d post-MPTP 13.0 + 1.3* 0.6 + 0.2 10.0 + 0.9* 1.1 + 0.4
30 d post-MPTP 21.6 + 6.1* 1.2 + 0.4 9.1 + 1.6* 1.9 + 0.6
Frontal Cortex Control 4.0 + 1.5 0.8 + 0.2 12.8 + 1.1 0.5 + 0.0
7 d post-MPTP 0.5 + 0.1* 1.4 + 0.2 6.7 + 1.5* 0.8 + 0.3
30 d post-MPTP 0.6 + 0.2* 4.2 + 1.3* 5.1 + 1.0* 2.5 + 1.0
Amygdala Control 9.2 + 2.5 0.7 + 0.1 11.9 + 0.8 1.2 + 0.2
7 d post-MPTP 4.8 + 1.0 0.8 + 0.3 8.6 + 1.6 1.9 + 0.8
30 d post-MPTP 3.5 + 1.0 2.1 + 1.0 6.8 + 1.3* 3.2 + 1.2
VME Control 5.2 + 0.8 1.1 + 0.0 23.7 + 0.7 1.1 + 0.0
7 d post-MPTP 5.1 + 1.2 1.8 + 0.6 20.7 + 4.9 2.8 + 1.2
30 d post-MPTP 3.8 + 2.0 4.6 + 1.4* 12.9 + 5.2 6.6 + 2.4
Raphe Nucleus Control 1.4 + 0.2 1.0 + 0.0 15.8 + 2.0 1.6 + 0.4
7 d post-MPTP 1.9 + 0.2 0.8 + 0.1 12.5 + 3.5 3.2 + 1.5
30 d post-MPTP 1.5 + 0.2 1.2 + 0.3 11.5 + 2.9 3.3 + 1.1
Concentrations are expressed as ng/mg of protein. Data are presented as mean ± SEM.
Abberviations: DA: dopamine; 5-HT: serotonine; VME: ventral mesencephalon.
DA turnover: ([DOPAC] + [HVA]) / [DA]; 5-HT turnover: [5-HIAA] / [5-HT]
* p<0.05 compared to the saline group.
Table 2.1: HPLC analysis of dopamine, serotonin, and their calculated turnover ratios in
the dorsal striatum, ventral striatum, frontal cortex, amygdala, ventral mesencephalon
(VME), and raphe nucleus from control, 7 days, and 30 days post-MPTP-lesioned mice
(n=5/group). Data are presented as mean ± SEM of monoamine concentrations (in
ng/mg of protein). Abbreviations: DA - dopamine, 5-HT – serotonin. Dopamine turnover
ratio was calculated as: ([DOPAC] + [HVA]) / [DA] and serotonin turnover ratio as: [5-
HIAA] / [5-HT]. The symbol “*” indicates statistically significant differences compared to
control group (p<0.05).
68
Fig. 2.6: Dopamine and serotonin levels in MPTP-lesioned mice. Twenty four hours
following behavioral testing, the dorsal striatum (dStr), ventral striatum (vStr), frontal
cortex (fCtx), amygdala, ventral mesencephalon (VME), and the raphe nucleus (RN)
tissue homogenates were analyzed for monoamine concentrations (n=5/group) using
HPLC. Data are presented as mean ± SEM. (A) Dopamine and (B) serotonin loss
relative to control mice in brain regions regulating associative memory and affective
behavior at 7 and 30 days post-MPTP-lesioning. The symbol “*
”
indicates statistically
significant differences compared to control group (p<0.05).
Among all six regions investigated, the dorsal striatum of saline-treated mice
contained the highest concentration of dopamine (141.3 ± 12.4ng dopamine/mg protein).
Here, acute MPTP-lesioning caused significant loss of dopamine that persisted for at
least 30 days (F
(2,14)
= 89.00; p<0.05). There was 95% depletion in mice at 7 days post-
MPTP (7.5 ± 1.6 ng dopamine/mg protein) and 86% depletion at 30 days post-MPTP-
lesioning (19.9 ± 5.2 ng dopamine/mg protein). The change in dopamine turnover ratio
did not reach significant difference at 7 days (1.1 ± 0.4) or 30 days post-MPTP-lesion
69
(1.4 ± 0.3) compared to controls (0.2 ± 0.0) (F
(2,14)
= 3.654; p>0.05). The ventral striatum
of saline-treated mice contained the second highest concentration of dopamine (91.0 ±
7.7 ng dopamine/mg protein). In this region MPTP-lesioning caused 86% depletion at 7
days post-MPTP-lesioning (13.0 ± 1.3 ng dopamine/mg protein) and 77% depletion at 30
days post-MPTP (21.6 ± 6.1 ng dopamine/mg of protein) (F
(2,14)
= 56.63; p<0.05).
However, differences in dopamine turnover ratio were not statistically significant (F
(2,14)
=
3.654; p>0.05), similar to dopamine turnover in the dorsal striatum.
In the frontal cortex of control mice dopamine concentration (4.0 ± 1.5 ng
dopamine/mg protein) was low compared to the striatum. Nonetheless, MPTP-lesioning
caused significant dopamine loss (88% depletion) at 7 days after MPTP (0.5 ± 0.1 ng
dopamine/mg protein) and at 30 days post-MPTP (0.6 ± 0.2 ng dopamine/mg protein;
86% depletion) (F
(2,14)
= 5.43; p<0.05). Furthermore, dopamine turnover ratio was
significantly increased at 7 (1.4 ± 0.2) and 30 days (4.2 ± 1.3) post-MPTP-lesioning,
compared to controls (0.8 ± 0.2) (F
(2,14)
= 5.863; p < 0.05).
In the amygdala of saline-treated mice dopamine levels (9.2 ± 2.5 ng
dopamine/mg protein) were about one-tenth of those in striatal regions. MPTP-induced
dopamine loss in amygdala did not reach statistical significance (F
(2,14)
= 3.132; p>0.05),
and dopamine turnover ratio remained similar between the groups (Table 2.1). Cell
bodies of dopamine and serotonin-producing neurons are located in VME and the raphe
nucleus, respectively. These two regions had low levels of dopamine in control mice
(Table 2.1). MPTP-lesioning did not cause significant dopamine loss in VME and the
raphe nucleus (Fig. 2.6A).
Serotonin loss was modest compared to dopamine loss following MPTP-
lesioning. In the dorsal striatum of control mice, serotonin was low (7.1 ± 0.8ng
serotonin/mg protein). Nonetheless, serotonin was significantly depleted at 7 days (3.7 ±
70
0.5ng serotonin/mg protein, 48% loss) and at 30 days post-MPTP-lesioning (3.3 ± 0.4ng
serotonin/mg protein, 64% loss) (F
(2,14)
= 13.10; p<0.05). The ventral striatum of control
mice contained twice as much serotonin compared to the dorsal striatum (16.4 ± 1.8ng
serotonin/mg protein). Here, the serotonin loss was moderate but still significant: 39%
depletion at 7 days and 44% at 30 days post-MPTP-lesioning respectively (10.0 ± 0.9
and 9.1 ± 1.6ng serotonin/mg protein; F
(2,14)
= 6.80; p<0.05). Serotonin turnover ratio in
dorsal and ventral striatum did not change significantly after MPTP-lesioning (Table 2.1).
Serotonin concentrations in the frontal cortex (12.8 ± 1.1ng serotonin/mg protein)
and amygdala (11.9 ± 0.7ng serotonin/mg protein) were similar in control mice. MPTP-
lesioning caused 48% depletion in the frontal cortex at 7 days (6.7 ± 1.5ng serotonin/mg
protein) and 60% at 30 days post MPTP-lesioning (5.1 ± 1.0ng serotonin/mg protein).
This decrease was statistically significant compared to controls (F
(2,14)
= 11.28; p<0.05).
There was no change in serotonin turnover ratio in the frontal cortex (Table 2.1).
Following MPTP-lesion in the amygdala, serotonin remained unchanged at 7 days post-
MPTP (8.5 ± 1.6ng serotonin/mg protein). However, serotonin was significantly depleted
at 30 days post-MPTP-lesioning (6.9 ± 1.3ng serotonin/mg protein) compared to control
(F
(2,14)
= 4.03; p<0.05).
The raphe nucleus and VME (containing SNpc, SNpr and VTA) had high average
serotonin concentrations in all mice (Table 2.1). These same brain regions had very low
levels of dopamine in all examined mice. However, MPTP-lesioning did not cause
significant depletion of serotonin in these regions.
TH-immunoreactivity in dorsal striatum and dopaminergic cell loss in SNpc
TH protein is often used as a functional biomarker of the integrity of
dopaminergic axons in the striatum (Jakowec et al 2004a; Petzinger et al 2007). There
71
was significant reduction of TH-immunoreactivity (TH-ir) in dorsal striatum of mice 7 and
30 days post-MPTP-lesioning (F
(2,9)
= 45.41; p<0.05). MPTP caused a 58% reduction of
TH-ir in the dorsal striatum examined at 7 days post-lesioning and 45% loss at 30 days
post-MPTP-lesioning.
The number of surviving TH-positive neurons in the SNpc was used as an
additional measure of the integrity of midbrain dopaminergic system 7 and 30 days post-
MPTP-lesioning. MPTP-lesioning caused 68% loss of TH-ir SNpc neurons in 7 days
post-MPTP and 66% loss at 30 days post-MPTP.
2.5. Discussion
The MPTP-lesioned mouse serves as a model of basal ganglia injury and
Parkinson’s disease. While the majority of studies focus specifically on motor deficits,
few studies have addressed affective behavior. The purpose of this study was to
examine non-motor behaviors (associative memory, conditioned fear, anxiety, and
depression) in the C57BL/6 mouse following acute lesioning with MPTP. We report
associative memory impairment measured by STFP evident only at 30 days post-MPTP.
In addition, mice had increased fear extinction at both 7 days and 30 days post-MPTP.
In contrast, there were no significant changes in anxiety (measured by hole-board and
light-dark preference tests), or depression (measured by sucrose preference and tail-
suspension tests). Dopamine and serotonin levels in brain homogenates were depleted
in the striatum, frontal cortex and amygdala.
Dopaminergic neurotransmission within the basal ganglia has been implicated in
cognitive processes, and specifically, in associative learning (Alcaro et al 2007).
However, only a few studies have examined memory function after basal ganglia injury
72
in rodent models. A previous study reported that MPTP-lesioned CD-1 mice had
impaired social memory and recognition behavior (Dluzen & Kreutzberg 1993). In our
study, the STFP test revealed impaired associative memory in mice only at 30 days
post-MPTP-lesion compared to saline-treated mice. At this time point, there was still an
86% depletion of dopamine and a 60% depletion of serotonin within the frontal cortex.
This is consistent with other studies using 6-OHDA-lesioning in rats where performance
in the STFP test depends on intact frontal cortex (Ross et al 2005) and basal forebrain
(Berger-Sweeney et al 2000). Taken together, these data support the importance of the
frontal cortex and the role of dopamine and serotonin in the associative learning
processes.
The fear conditioning response is mediated by the basolateral amygdala,
hippocampus, medial prefrontal cortex, and nucleus accumbens (Davis & Whalen 2001;
Helmstetter 1992; LeDoux 2000; Maren & Quirk 2004). The extinction of conditioned fear
is a progressive decrease of the fear response generated by repeated presentation of
the conditioned stimulus (tone) without any unconditioned stimulus (foot-shock).
Changes in the extinction of conditioned fear can be influenced by either glutamate or
dopamine neurotransmission particularly in the frontal cortex and amygdala (Falls et al
1992; Guarraci et al 1999; Ledgerwood et al 2003; Walker et al 2002). For example,
dopamine D1 and D2 receptor antagonists targeting the amygdala can lead to
potentiated extinction of fear response (Greba et al 2001; Greba & Kokkinidis 2000;
Nader & LeDoux 1999; Ponnusamy et al 2005). In our study, MPTP-lesioning had no
effect on the acquisition of the fear response; instead we observed increased fear
extinction at both early and late time points after MPTP-lesioning. Interestingly, a recent
human study reported decrease of the startle response to aversive stimuli in patients
with PD and this behavior was linked to dopamine dysfunction in the amygdala and
73
frontal cortex (Bowers et al 2006). Although dopamine loss in the amygdala did not
reach statistical significance in our MPTP-lesioned mice, altered cortical input to the
amygdala could be responsible for observed behavioral response. It is possible that
significant loss of dopamine in the frontal cortex in mice at 7 and 30 days post-MPTP
could be involved in this process. It is know that GABAergic interneurons surrounding
the basolateral amygdala receive cortical and mesolimbic dopaminergic input (Marowsky
et al 2005). It is hypothesized that modulation of dopamine gate in these neurons can
modulate stress-induced behavioral responses (Bowers et al 2006; Marowsky et al
2005). Dopamine loss in the frontal cortex, which we observed in MPTP-lesioned mice,
could cause disinhibition of these interneurons and block the output of the amygdala in
response to aversive stimuli. This mechanism could prevent the normal fear induced
freezing response, like the one we observed in saline-treated mice.
Dopamine, serotonin and other neurotransmitter system perturbations are
involved in anxiety disorders and may account for the clinical anxiety seen in more than
40% of patients with PD (Walsh & Bennett 2001; Wood & Toth 2001). In our study,
using light-dark exploration and the hole-board tests we found no increase in anxiety in
mice at either 7 or 30 days post-MPTP-lesioning. These results are in agreement with
others who also showed no difference in anxiety using the light-dark preference test 7
days after MPTP-lesioning in the mouse (Rousselet et al 2003). Depression is the most
common co-morbid disorder affecting up to 45% of patients with PD (Slaughter et al
2001). We examined depression in our model using both the tail suspension and
sucrose preference tests, since both may be influenced by serotonin dysfunction (Jones
& Lucki 2005; Lira et al 2003; Mayorga et al 2001; Steru et al 1985). We did not observe
increased depression in MPTP-lesioned mice using these tests. Interestingly, a recent
study using the bilateral 6-OHDA-lesioned rat reported an increase in depression-like
74
behavior that was measured using the forced swim test, with no changes in serotonin
levels (Branchi et al 2008). It is possible that lesioning paradigm, time after lesion and
species used could underlie differences in observed behaviors in these two animal
models.
Lesioning of the dopaminergic system, using 6-OHDA or MPTP, also leads to
perturbations of the serotonergic system. However, the extent and nature of these
perturbations depends on a number of factors including animal age, toxin used, lesioning
regimen, and lesion severity. For example, rats and mice lesioned with 6-OHDA in early
postnatal life develop serotonergic hyper-innervation within the striatum and frontal
cortex and elevated levels of serotonin (Avale et al 2004; Berger et al 1985; Snyder et al
1986; Yamazoe et al 2001). This is in contrast to lesioning in adults where serotonin
levels remain unchanged (Branchi et al 2008; Snyder et al 1986; Stachowiak et al 1986).
On the other hand, MPTP-lesioning in adult animals causes a significant decrease of
striatal and extra-striatal serotonin levels. For example, in the nonhuman primate,
MPTP-lesioning causes decreased levels of serotonin in multiple brain regions including
the caudate nucleus, putamen, nucleus accumbens, hypothalamus and cerebral cortex
(Frechilla et al 2001; Perez-Otano et al 1991b; Pifl et al 1991; Russ et al 1991). In our
studies, we found significant depletion of serotonin following MPTP-lesioning in the
mouse, a similar effect seen by others (Rousselet et al 2003) but not all (Sedelis et al
2000). Despite serotonin depletion in brain regions important for affective behavior, our
MPTP-lesioning mouse did not show significant changes in anxiety and depression. The
lack of behavioral effect could be explained by (i) the level of serotonin depletion may
not be severe enough to manifest elevated anxiety, or (ii) the affected neurotransmitter
systems may compensate to overcome perturbation. The serotonergic system following
MPTP-lesioning may compensate in an analogous fashion to that of the dopaminergic
75
system where studies in our lab have shown recovery of dopamine function due to
increased evoked release of dopamine and altered dopamine receptor expression
(Petzinger et al 2007). On the other hand, studies in PD patients have shown neuronal
loss in the dorsal raphe nucleus which could cause mood disorders (Agid & Blin 1987;
Paulus & Jellinger 1991; Scatton et al 1983). However, these abnormalities in humans
are thought to develop at later stages of the disease, following dysfunction of
dopaminergic system (Dauer & Przedborski 2003). With this in mind, there is a
possibility that behavioral changes in our acute MPTP-lesioned mouse may develop at
later time points after treatment.
The MPTP-lesioned mouse is commonly used as an animal model of PD.
Specific motor functions such as skilled forepaw use, balance, and coordination have
been consistently reported to be impaired following MPTP-lesioning (Meredith & Kang
2006; Meredith et al 2008; Rozas et al 1998; Sedelis et al 2000; Tillerson et al 2002a;
Tillerson & Miller 2003). The behavioral tests used in our study do not rely on any of
these specific motor skills. For example, control and MPTP-lesioned mice showed
similar levels of ambulatory activity in the hole-board and light-dark preference tests.
We therefore conclude that MPTP-lesioned mice in the present study did not exhibit
obvious impairment of spontaneous locomotor activity that could influence their
performance in affective behavior tests.
In conclusion, our data showed impairment in associative memory at 30 days
and increased fear extinction at both 7 and 30 days post-MPTP-lesioning, but no
significant increase in depression or anxiety. Impairments in memory and fear
conditioning were accompanied by severe depletion in dopamine and serotonin levels in
the amygdala, frontal cortex, and striatum. It is possible that the emergence of
depression and anxiety in this mouse model depends upon greater serotonin loss in
76
critical brain regions. The findings from this study suggest that mood disorders in
patients with may not develop before an extensive damage in multiple brain regions
occurs.
77
CHAPTER 3:
EXERCISE ELEVATES DOPAMINE D2 RECEPTOR IN A MOUSE MODEL OF
PARKINSON’S DISEASE: IN VIVO IMAGING WITH [
18
F]FALLYPRIDE
2
3.1. Abstract
The purpose of the current study was to examine changes in dopamine D2
receptor (DA-D2R) expression within the basal ganglia of MPTP mice subjected to
intensive treadmill exercise. Using western immunoblotting analysis of
synaptoneurosomes and in vivo positron emission tomography (PET) imaging employing
the DA-D2R specific ligand [
18
F]fallypride, we found that high intensity treadmill exercise
led to an increase in striatal DA-D2R expression that was most pronounced in MPTP
compared to saline treated mice. Exercise-induced changes in the DA-D2R in the
dopamine-depleted basal ganglia are consistent with the potential role of this receptor in
modulating medium spiny neurons (MSNs) function and behavioral recovery.
Importantly, findings from this study support the rationale for using PET-imaging with
[
18
F]fallypride to examine DA-D2R changes in individuals with PD undergoing high
intensity treadmill training.
3.2. Introduction
Exercise improves motor performance in patients with Parkinson’s disease (PD)
(Bergen et al 2002; Comella et al 1994b; Schenkman et al 2008). While our
understanding of the processes that may underlie this behavioral improvement is limited,
animal models, such as the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
2
Results of this chapter are accepted for publication in the Movement Disorders journal (Vuckovic et al
2010a).
78
mouse, provide a critical tool to investigate the molecular mechanisms of exercise-
induced improvement in motor behavior (Fisher et al 2004; Petzinger et al 2007;
Pothakos et al 2009). The dopamine D1 and D2 receptors (DA-D1R and DA-D2R) are
the primary targets of dopamine on medium spiny neurons (MSNs) within the striatum
and modulate physiological properties and cellular signaling. Specifically, the DA-D2R is
known to play a major role in long-term depression (LTD), a form of synaptic plasticity
that involves integration of glutamatergic and dopaminergic synaptic neurotransmission
at the level of the MSNs leading to the encoding of motor function within the dorsolateral
striatum. Given the role of the DA-D2R in motor control and learning, we sought to
examine whether exercise enhanced improvement in motor function may be due in part
to an increase in striatal DA-D2R expression.
Positron emission tomography (PET) with DA-D2R radiotracers offers the ability
to carry out longitudinal studies on the effect of exercise in humans. Previous studies on
the effects of aerobic exercise have attempted to measure dopamine release in normal
individuals (Wang et al 2000). However, no change in the binding of [
11
C]raclopride was
observed leading the authors to suggest that little change in dopamine levels occurred.
[
18
F]fallypride is a high affinity PET-imaging ligand specific for both DA-D2R and DA-
D3R, and unlike the more commonly used [
11
C]raclopride, it is not readily displaced by
baseline levels of endogenous dopamine
7-10
. This was confirmed by reserpine
pretreatment of animals (to deplete endogenous dopamine) which had no effect on
[
18
F]fallypride binding
9,25
, but significantly increased [
11
C]-raclopride binding (Ginovart et
al 1997). This increase in [
11
C]-raclopride binding was attributed to a change in apparent
binding affinity (K
d
) rather than receptor number (B
max
).
Since the binding potential (BP) of [
18
F]-fallypride was resistant to changes due to
depletion of dopamine suggesting little effect on its K
d
or B
max
at baseline and depleted
79
state, we considered the [
18
F]-fallypride to investigate our hypothesis of a potential
increase in DA-D2R expression in the MPTP mouse model with and without exercise
(Christian et al 2000; Honer et al 2004; Mukherjee et al 2001; Mukherjee et al 1999).
Furthermore, in order to provide support to PET measures, we used the complementary
technique of western immunoblotting to measure changes in DA-D2R protein expression
in the same animals. We report here the effects of exercise on DA-D2R expression and
[
18
F]fallypride in groups of mice treated with either saline or MPTP.
3.3. Materials and methods
Animals, treatment groups, and MPTP administration
Male C57BL/6 mice 8 weeks old (Charles River Laboratories, Wilmington, MA)
were group-housed in a temperature-controlled room under 12h light/12h dark cycle. All
procedures were performed in accordance with the NIH Guide for the Care and Use of
Laboratory Animals as approved by the USC IACUC. A total of 164 mice were used in
four treatment groups: (1) saline (n = 42), (2) saline plus exercise (n = 55), (3) MPTP (n
= 57), and (4) MPTP plus exercise (n = 42). For lesioning, mice received 4
intraperitoneal injections of 20 mg/kg MPTP (free-base; Sigma-Aldrich, St. Louis, MO)
dissolved in 0.9% saline, at 2h intervals or 4 intraperitoneal injections of 0.1ml 0.9%
NaCl as control. Lesioning was validated by HPLC analysis of striatal dopamine levels.
At 10 days post-MPTP administration, there was 82.2% dopamine depletion in MPTP
mice (48.0 ± 8.4 ng/mg of protein) compared to saline mice (269.5 ± 24.9 ng/mg of
protein). At the end of the study, there was no significant difference in striatal dopamine
levels between MPTP plus exercise mice (69.8 ± 11.7 ng/mg of protein) compared to
MPTP (77.9 ± 12.0 ng/mg of protein). However, there was a significant increase of
80
striatal dopamine in saline plus exercise mice (315.2 ± 9.0 ng/mg of protein) compared
to saline (246.9 ± 19.8 ng/mg of protein) (F
(3,16)
= 7.78; p<0.05).
Treadmill exercise
Exercise started 5 days after lesioning. Mice from the two exercise groups (saline
plus exercise and MPTP plus exercise) were trained to run on a 100cm motorized
treadmill (Exer 6M, Columbus Instruments, OH) at incremental speeds for 6 weeks (5
days /week) to reach duration of 60 min/day and speed of 18-20 m/min (Fisher et al
2004; Petzinger et al 2007).
Magnetic resonance imaging
A three-dimensional volumetric T1-weighted magnetic resonance (MR) image of
the mouse brain was obtained on a 7 Tesla micro-MRI system (Bruker Biospin, Billerica,
MA). Parameters of image acquisition were as follows: TE = 46.1ms, TR = 6292.5 ms,
0.4mm slice thickness, 0.45mm inter-slice thickness, 128 x 128 x 128 matrix size.
Radiochemistry
The synthesis of [
18
F]fallypride was carried out as previously described by
nucleophilic substitution reaction of the tosyl precursor with [
18
F] using a custom-made
radiochemistry apparatus (Mukherjee et al 1999). Purification of the reaction mixture was
performed by reverse-phase high performance liquid chromatographic (HPLC)
separation on a C8(2) Phenomenex Luna column using acetonitrile and sodium
phosphate buffer as mobile phase (55:45)
. UV absorbance was measured at
wavelength 254nm and AUFS 0.05. Radioactive peak (retention time 17 min)
corresponding to [
18
F]fallypride, was collected and solvent removed on a rotary
81
evaporator. The final product was tested for pyrogenicity, sterility, pH, and removal of
organic solvents by gas chromatography. Specific activity and radiochemical purity was
assessed with a Waters HPLC system using a C8(2) Phenomenex Luna analytical
column. Specific activity was in the range of 3,000 – 12,000 Ci/mmol.
PET measurements and image analysis
Twenty mice were used for PET imaging (n=6 saline; n=3 saline plus exercise;
n=5 MPTP; and n=6 MPTP plus exercise). Scans were acquired with a Concorde
microPET R4 scanner (CTI Concorde Microsystems, Knoxville, TN) with a 60 min list
mode acquisition protocol after 20 min transmission scan for attenuation correction with
a
68
Ge source. [
18
F]fallypride (10.92-11.28MBq) was injected via the tail vein (single
bolus) at the start of the emission scan. Mice were anesthetized with 2% isofluorane and
98% oxygen. The dynamic list mode data was sorted to sinograms with 26 frames (6x20
sec, 4x40 sec, 6x1 min, and 10x5 min), and reconstructed by 2 iterations of OSEM
(ordered subsets expectation maximization) followed by 18 iterations of the MAP
(maximum a posteriori) reconstruction algorithm (Qi et al 1998). Reconstructed images
were cropped to contain the head and linearly interpolated in the Z-direction to produce
a 128x128x63 image with isotropic 0.4mmx0.4mmx0.4mm voxels. High resolution
binding potential (BP) images of the striatum were computed from the reconstructed
dynamic images using a multi-linear tissue reference model (Ichise et al 2002) and
Logan plots (Logan et al 1996) with high activity in the striatum and very low activity in
the cerebellum, which was used as a reference region. Anatomical regions of interest
(dorsal striatum and cerebellum) were manually defined in both hemispheres in PET
images co-registered with MRI using Rview (Studholme et al 1997). Quantification of
specific binding of [
18
F]fallypride in the mouse striatum was performed using the binding
82
potential (BP) value that provides a measure of the ratio of specific to non-specific
radioligand binding at equilibrium (Lammertsma & Hume 1996; Mintun et al 1984). To
demonstrate binding specificity in the striatum, 4 mice were harvested 60 minutes after
ligand injection, brains quickly frozen in liquid nitrogen, sectioned at 30-micron thickness,
and sections apposed to a phospho-imager (Typhoon 9200, GE Healthcare Inc.,
Piscataway, NJ) (Figure 3.1). Studies have shown that [
18
F]fallypride binds specifically to
the DA-D2R and since very little DA-D3R is in the striatum, binding indicates DA-D2R
occupancy (Christian et al 2000; Honer et al 2004; Mukherjee et al 2001; Mukherjee et al
1999).
Figure 3.1: [
18
F]fallypride shows high biding specificity to the mouse striatum. The left
panel shows an anatomical rendering of the coronal section at approximate level bregma
0.20. The right panel shows a representative autoradiograph with intensive labeling
corresponding to the striatum.
Tissue collection for HPLC and protein analysis
At the end of the study brains were quickly removed and dorsal striatum
dissected fresh corresponding to anatomical regions from bregma 1.2 to 0.6 with the
corpus callosum as dorsal border, the lateral aspect of the corpus callosum as lateral
border, and above the anterior commissure as the ventral border (Paxinos & Franklin
2001).
83
HPLC analysis of dopamine and its metabolites
Dopamine levels in striatal homogenates (n = 4 per group) were determined by
HPLC with electrochemical detection as previously described (Petzinger et al 2007). The
system consisted of an ESA auto-sampler (ESA Inc., Chelmsford, MA) equipped with a
150 x 3.2 mm reverse phase C-18 column (3µm diameter) and a CoulArray 5600A (ESA
Inc, Chelmsford, MA), equipped with a 4-channel analytical cell with potentials set at -
100mV, -30mV, 220mV and 350mV.
Western immunoblot analysis
Synaptoneurosome preparations were made from fresh striatal tissue (n=8 per
group) following published methods (Johnson et al 1997). Tissue blocks were
immediately placed into ice-cold homogenization buffer consisting of 10mM HEPES, pH
7.4, 1mM EDTA, 0.25mM dithiothreitol, 0.35M sucrose and 1:100 vol/vol protease
inhibitor cocktail sets I and III and phosphatase inhibitor cocktail set II (Calbiochem, La
Jolla, CA). Tissue was homogenized using Teflon-glass mechanical tissue grinder with
0.25mm clearance (Wheaton, Millville, NJ) by applying 4 strokes at 1000 rpm. Cell nuclei
were separated by centrifugation at 1000 x g for 10 min at 4°C. The supernatant
containing cytosol and small cellular organelles was forcefully pass through a series of
nylon mesh screens with decreasing pore size 100/78/25 and 5µm (Small Parts Inc.,
Miramar, FL) using a syringe. Final filtrate was re-suspended in 3 volumes of
homogenization buffer without sucrose. Synaptoneurosomes and other cellular particles
were separated from small debris by centrifugation at 2000xg for 15min at 4°C. Pellet
was re-suspended in 50µL of 10mM HEPES buffer pH 7.4, aliquoted into 40µg total
protein samples and kept frozen at -80°C until use. Total protein concentration in each
sample was determined from a small aliquot (<10µL) using Pierce BCA Protein Assay
84
Kit (Pierce, Rockford, IL) according to the manufacturer’s protocol following the
microplate procedure.
The relative expression of proteins for DA-D1R (50kDa), DA-D2R (65kDa),
tyrosine hydroxylase (68kDa), and alpha-tubulin (50kDa) (as loading control) was
analyzed by western blot (Laemmli 1970) using commercially available primary
antibodies (rabbit polyclonal antibodies against DA-DR1 and DA-DR2 and mouse
monoclonal against tubulin, Millipore, Temecula, CA) specific for each protein.
Synaptoneurosomal samples containing 40µg total protein were denatured by boiling at
95°C for 5min in 2xSDS sample buffer consisting of 250mM Tris pH 6.8, 40% glycerol,
4% SDS, 4% beta-mercaptoethanol, and 0.2% bromphenol blue. Synaptic proteins were
separated on 12% Tris-Glycine polyacrylamide gels (Lonza, Rockland, ME) using
constant voltage of 150V in electrophoresis buffer consisting of 25mM Tris pH 8.3,
192mM glycine and 0.1% SDS. Proteins were transferred to nitrocellulose membranes
using constant current of 50mA for 18h in transfer buffer containing 48mM Tris pH 8.3,
39mM glycine, 20% methanol and 0.037% SDS. Membranes were first incubated in 2x
blocking buffer for infrared fluorescence immunoblot detection (Rockland, Gilbertsville,
PA) and then exposed to primary antibodies (1:200) for 24h at 4ºC. After washing,
membranes were exposed to secondary antibodies conjugated to infra-red fluorescent
dye: IRDye-800 affinity purified goat anti-mouse or IRDye-680 affinity purified goat anti-
rabbit antibodies (Rockland, Gilbertsville, PA). Fluorescent signal was detected by
scanning the membranes in LI-COR Odyssey near infrared imaging platform (LI-COR
Biotechnology, Lincoln, NE). Quantification of fluorescent signal from specific bands was
performed using Odyssey 2.1 software (LI-COR Biotechnology, Lincoln, NE).
85
Statistical analysis
The differences between experimental groups in BP of [
18
F]fallypride, DA-D1R
and DA-D2R protein levels were analyzed using two-way analysis of variance (ANOVA)
with treatment as between subject factor (saline vs. MPTP), and exercise as within
subject factor (no exercise vs. exercise). For maximal treadmill speed test, time was
used as between subject factor (week 1, 2, etc.) and treatment was used as within
subject factor (saline vs. MPTP). The Bonferroni post hoc test was used to correct for
multiple comparisons when assessing significance of interest. Significance level was set
to p<0.05. To explore the practical significance of group differences, an estimate of the
magnitude of the differences between groups was calculated using effect size (ES) (ES
= Mean
Group 1
– Mean
Group 2
/SD
pooled
. The ES is a value that reflects the impact of a
treatment within a population of interest and is reported according to established criteria
as small (< 0.41), medium (0.41 - 0.70), or large (> 0.70) (Thomas et al 1991). Analysis
was performed using Prism5 for Windows (Graph Pad, San Diego, CA).
3.4. Results
High intensity treadmill exercise improves motor behavior in MPTP-lesioned mice
Prior to MPTP-lesioning and start of exercise, average baseline velocities of all
mice in two exercise groups were similar (saline plus exercise: 11.7 ± 1.1 m/min, and
MPTP plus exercise: 11.2 ± 1.1 m/min). Daily exercise for 6 weeks improved maximal
treadmill velocities in both exercise groups with the saline plus exercise mice displaying
significantly greater maximal velocity compared to the MPTP plus exercise mice in
weeks 1 through 4 (Fig. 3.2). However, MPTP plus exercise mice had similar maximal
treadmill speeds as saline plus exercise mice in weeks 5 (MPTP plus exercise: 17.2 ±
3.6 m/min and saline plus exercise: 22.0 ± 1.5 m/min) and 6 (19.2 ± 1.2 m/min and 22.2
86
± 0.9 m/min, respectively). As previously reported, MPTP-lesioned mice that did not
undergo treadmill training displayed no spontaneous recovery of motor behavior with
their maximal velocity of 7.5 m/min at the end of the 6-week exercise period (Fisher et al
2004).
Figure 3.2: Exercise improves motor behavior in the MPTP mouse. The maximum
running speed of saline (n=12) and MPTP (n=12) mice on the motorized treadmill was
tested at the end of each week. The baseline treadmill velocities were measured prior to
MPTP lesioning. By the end of the running period there was no difference in velocity
between saline and MPTP mice. Data were analyzed by two-way ANOVA with repeated
measures; the symbol “ * ” represents significance level p<0.05. Significant differences in
maximal treadmill velocity were seen at weeks 1 through 4.
High intensity treadmill exercise increases striatal DA-D2R but not DA-D1R protein
High intensity treadmill exercise differentially affected DA-D2R and DA-D1R
levels in synaptoneurosomal preparations from the dorsal striatum as shown by western
blot analysis (Fig. 3.3). MPTP plus exercise mice had 48.8% increase in striatal DA-D2R
compared to MPTP mice (Fig. 3.3 B). There was a significant effect of exercise (F
(1,8)
=
25.7; p<0.05), and significant interaction between exercise and MPTP lesioning on DA-
D2R protein level (F
(1,8)
= 6.0; p<0.05). Conversely, there was no exercise effect on DA-
D1R protein levels between the groups (Fig. 3.3A) (F
(1,8)
= 0.1, p = 0.78). MPTP lesioning
alone did not significantly alter either DA-D2R (F
(1,8)
= 0.0; p = 0.88) or DA-D1R
87
expression (F
(1,8)
= 0.0; p = 0.92). In addition, two different protein markers of midbrain
dopaminergic fibers integrity, tyrosine hydroxylase (TH; Fig. 3.3C) and dopamine
transporter (DAT; Fig. 3.3D), showed that MPTP significantly decreased striatal TH
protein (F
(1,8)
= 757.3; p<0.05) and DAT expression (F
(1,8)
= 218.0; p<0.05).
Figure 3.3: Exercise selectively up-regulates DA-D2R but not DA-D1R striatal protein.
Panel (A) shows western immunoblot analysis of synaptoneurosome preparations from
the dorsal striatum for DA-D1R protein. There was no statistically significant difference
between treatment groups. Panel (B) shows western immunoblot analysis of
synaptoneurosome preparations from the dorsal striatum for DA-D2R protein. MPTP
plus exercise mice showed elevated DA-D2R protein compared to MPTP mice. (C)
Analysis of tyrosine-hydroxylase (TH) protein, and (D) dopamine transporter (DAT)
protein (markers of midbrain dopaminergic terminals) in synaptoneurosome preparations
from four different groups. MPTP treatment significantly decreased levels of TH and
DAT proteins in dorsal striatum. These data were generated in three separate
experiments, each consisting from pooled tissue (n=8 brains/per group). Results are
shown as mean ± S.E.M. Symbols “ * ” and “#” represent significance level p < 0.05, two-
way ANOVA with Bonferroni correction.
88
High intensity treadmill exercise increases striatal [
18
F]fallypride binding potential (BP)
While western immunoblotting analysis of receptor protein expression measured
total antibody epitopes (both surface and internal cellular stores), in vivo PET-imaging
with the high affinity DA-D2R specific radioligand [
18
F]fallypride can delineate the effects
of exercise on availability of DA-D2R to bind ligand (Fig. 3.4A). Statistical analysis
revealed that there was a significant effect of exercise (F
(1,16)
= 12.3; p<0.05) as well as
MPTP lesion (F
(1,16)
= 160.3; p<0.05) with no significant interaction between MPTP and
exercise (F
(1,16)
= 3.5; p = 0.07) on [
18
F]fallypride BP. The Bonferroni post-hoc analysis
showed significant difference in BP values between MPTP and MPTP plus exercise
mice (t = 1.1, Df = 1, 16; p<0.01), and no significant difference between saline and saline
plus exercise mice (t = 4.1, Df = 1, 16; p>0.05). Specifically, MPTP plus exercise mice
had a 73.1% increase in [
18
F]fallypride BP compared to MPTP mice (average BP values
for MPTP plus exercise: 7.1 ± 0.7; average BP values for MPTP mice: 4.1 ± 0.3) (Fig.
3.4B). In addition, saline plus exercise mice had an 8.2% increase in [
18
F]fallypride BP
(13.2 ± 1.0) compared to saline mice (12.2 ± 0.3). Consistent with these findings Effect
Size calculations revealed a larger exercise effect between MPTP groups (ES = 2.61)
than that observed between the saline groups (ES = 0.94).
89
Figure 3.4: Exercise selectively increases [
18
F]fallypride binding potential (BP) in the
striatum of MPTP mice. Panel (A) shows [
18
F]fallypride BP representative images in the
coronal orientation (left side) and horizontal orientation (right side). The scale bar to the
right shows relative BP intensity with high in red and low in blue. Panel (B) graphically
depicts the BP data showing that MPTP reduced [
18
F]fallypride BP, while exercise
increased [
18
F]fallypride BP in both saline and MPTP mice. Results are shown as mean
± S.E.M. The symbol “#” represents significance level of MPTP effect (p < 0.05), and “*”
represents the effect of exercise in MPTP groups as shown by two-way ANOVA with
Bonferroni post-hoc correction (p<0.01).
90
3.5. Discussion
This study demonstrates that high intensity treadmill exercise leads to an
increase in [
18
F]fallypride BP (DA-D2R availability) in the striatum of MPTP treated mice.
Conversely, there was no significant change in total striatal dopamine levels between
MPTP plus exercise compared to MPTP no exercise mice. [
18
F]fallypride is a highly
selective DA-D2/D3R antagonist whose BP reflects an in vivo measure of available
receptors (B
max
)/binding affinity (K
d
). Since DA-D2Rs are the predominant dopamine
receptor subtype within dorsal striatum, an exercise-induced increase in [
18
F]fallypride
BP represents an increase in DA-D2R number and is supported by an increase in
protein expression using western immunoblotting and our previous studies showing an
increase in striatal DA-D2R mRNA transcript expression using in situ hybridization
histochemistry (Fisher et al 2004). This interpretation of BP elevation is further
supported by the fact that displacement of [
18
F]fallypride by dopamine is not likely to
occur in the MPTP mouse, since dopamine levels remain low (Cropley et al 2008).
Hence changes in apparent binding affinity (K
d
) are negligible and are unlikely to have
an effect on BP. The enhanced effect of exercise in MPTP mice may reflect an attempt
of the injured brain to optimize dopaminergic neurotransmission through increased
receptor number while dopamine levels remain depleted. Increased responsiveness of
MPTP mice to exercise reveals a greater potential of the injured versus the intact brain
to undergo neuroplasticity, which may not be essential when striatal circuitry is intact.
The fact that dopamine levels do not change significantly with exercise in MPTP mice
suggests that compensatory changes in DA-D2R are critical for exercise related
improved motor performance.
Using PET-imaging, we observed a decrease in DA-D2R BP after MPTP
lesioning relative to saline treated mice. This was in contrast to western immunoblotting
91
in which no change in DA-D2R protein expression was observed. The DA-D2R exists in
a dynamic equilibrium between surface and intra-cellular compartments, with the latter
not generally available to binding to PET radioligands. In the dopamine-depleted state,
compensatory mechanisms may lead to changes in the intracellular pool for DA-D2R,
which may be unavailable for [
18
F]fallypride binding but yet available for detection in
western immunoblotting.
Unlike our findings, a compensatory increase in the DA-D2R has been reported
in individuals with PD and after administration of MPTP in nonhuman primates and cats,
or 6-OHDA in rats (Hurley & Jenner 2006). In the literature, the loss of DA-D2Rs is
reportedly due to the degeneration of dopaminergic neurons, whereas the increase in
DA-D2Rs results from increased expression on remaining dopaminergic terminals,
and/or increased synthesis within striatopallidal neurons or cholinergic interneurons.
This discrepancy between our PET study, and those of the literature, may be due to
differences in the severity of the lesion between studies (Falardeau et al 1988).
Specifically, the loss of a greater number of pre-synaptic DA-D2Rs through MPTP-
induced cell loss may be sufficient to offset any post-synaptic compensatory changes
induced by the lesion alone. Alternatively, our inability to observe an increase in DA-D2R
BP and expression levels in MPTP (non-exercise) mice may be due to a modest
recovery of dopamine levels at the end of the study (82% dopamine depletion at 10 days
versus 68% depletion at 42 days post-lesion). However, this is unlikely since the MPTP
plus exercise mice, that also displayed a small recovery of dopamine (not significantly
different from the MPTP no exercise mice) had increase of DA-D2R BP.
The majority of DA-D1Rs and DA-D2Rs are expressed on dendritic spines of
MSNs with additional dopamine receptors expressed on cholinergic interneurons as well
as the terminals of glutamatergic and dopaminergic neurons originating from the cortex
92
(or thalamus) and substantia nigra pars compacta, respectively (Smith & Villalba 2008).
A major role of dopamine within the striatum is to modulate cortico- or thalamostriatal
glutamatergic neurotransmission within the MSN. As such, glutamatergic
neurotransmission is enhanced through DA-D1Rs and diminished through DA-D2Rs
(Cepeda 1993; Levine et al 1996; Umemiya & Raymond 1997). Under conditions of
dopamine depletion spines and synaptic connections are selectively lost on DA-D2R
expressing neurons of the indirect pathway. This loss is accompanied with a hyper-
excitability state within the MSNs and observed as increased post-synaptic current
amplitude and frequency due to increased glutamatergic corticostriatal
neurotransmission (Day et al 2006; Hernandez-Echeagaray et al 2004; Surmeier et al
2007; Vanleeuwen et al 2009). In animal models of PD, this increased glutamatergic
drive correlates with parkinsonian-like behavior (Calabresi et al 1993). Attenuation of this
hyper-excitable state through the application of dopamine or quinpirole (a selective DA-
D2R agonist) leads to reversal of parkinsonian motor deficits (Ballion et al 2009;
Calabresi et al 1997c). In light of these reports and our findings, we hypothesize that
the benefits of high intensity exercise are to enhance dopaminergic signaling through
increased DA-D2R expression in the indirect pathway (but not the DA-D1R of the direct
pathway) and to improve motor function through suppression of glutamatergic
excitability.
The primary conclusion of our study is that exercise in the form of intensive
treadmill running facilitates neuroplasticity through increased expression of striatal DA-
D2Rs and that this process is most evident in the injured versus the intact brain. In light
of these present findings, a non-invasive PET-imaging approach with [
18
F]fallypride can
be used to investigate whether intensive treadmill exercise also leads to changes in the
DA-D2R in individuals with PD. Our study highlights the value of preclinical research in
93
animal models of dopamine depletion and the importance of translational research for
providing both rationale and insight towards understanding imaging and exercise studies
in individuals with PD.
94
CHAPTER 4:
TREADMILL EXERCISE REVERSES MPTP SPINE LOSS IN STRIATAL MEDIUM
SPINY NEURONS
3
4.1. Abstract
Treadmill exercise can improve motor function following brain injury, however the
neuronal mechanisms underlying this effect are not completely understood. We used
Golgi-Cox technique and electron microscopy to investigate the effect of high intensity
treadmill running on morphology of striatal medium spiny neurons (MSNs) in the 1-
methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated mouse model of basal
ganglia injury. Our results showed recovery of dendritic spine density on MSNs in
MPTP-lesioned plus exercise mice compared to sedentary MPTP mice. Exercise also
increased spine density in control mice. Electron microscopic analysis showed a
significant increase of synapse number in the dorsal striatum in lesioned and control
mice following treadmill exercise. Furthermore, ultrastructural analysis of dendritic spine
area showed no change between the treatment groups. Taken together, studies in this
report show that behavioral activity, such as motor training, promotes spine and synapse
formation in injured basal ganglia in mice.
Keywords: basal ganglia, dopamine, glutamate, spine density, neuroplasticity, electron
microscopy
3
Results of this chapter are under revision for publication in the journal Neuro Report (Vuckovic et al
2010b).
95
4.2. Introduction
Previous studies showed that enriched environment, motor training and learning
of a new motor skill promotes dendritic spine formation on neurons from the motor
cortex, cerebellum and striatum in healthy animals (Black et al 1990; Comery et al 1996;
Rosenzweig & Bennett 1996; Xu et al 2009a; Yang et al 2009). However, much less is
known whether similar processes can be initiated in an injured brain, and in particular in
the injured basal ganglia. Studies using animal models of stroke reported alternations in
dendritic morphology that were implicated in recovery of motor function following brain
injury (Biernaskie & Corbett 2001; Bury et al 2000; Jones & Schallert 1994; Takamatsu
et al 2010; Xu et al 2009b). Here we show recovery of dendritic spine density and
synapse formation following treadmill training in MPTP mouse model of basal ganglia
and dopamine depletion.
The striatum is the main input nuclei of the basal ganglia and the major target of
dopaminergic projections from the substantia nigra pars compacta (SNpc). The principal
neurons in the striatum are medium spiny neurons (MSNs), which receive two types of
input through their dendritic spines – excitatory glutamatergic neurotransmission from
the motor cortex and thalamus, and modulatory dopaminergic projections from SNpc.
Severe loss of dopamine in the striatum is accompanied by dramatic spine loss on
MSNs in humans affected with Parkinson’s disease (PD) (Stephens et al 2005; Zaja-
Milatovic et al 2005). Similar findings have been reported for MPTP-treated monkeys
(Villalba et al 2009) and rodent models of basal ganglia injury (Ingham et al 1998). Spine
loss on MSNs contributes to functional impairments in glutamate corticostriatal and
thalamo-striatal neurotransmission. Recent findings in MPTP treated monkeys showed
that after the initial spine loss, remaining spines on the striatal MSNs go through
96
complex ultra-structural changes in response to increased glutamate synaptic activity
(Smith et al 2009b).
Enriched environment enhances spine formation in rodents (Rosenzweig &
Bennett 1996). Experience dependent synaptic plasticity initiates growth, retraction, or
structural reorganization of existing or new spines and synapses. In the hippocampus,
changes in shape and size of dendritic spines are proposed to be a cellular substrate for
memory formation and storage. Numerous studies have shown that in hippocampus,
induction of LTP causes changes in spine density (Harris & Kater 1994; Kasai et al
2003; Segal 2005; Yuste & Bonhoeffer 2001). Learning of new motor skill is associated
with formation of a new set of spines in cortical pyramidal neurons, followed by loss of
different set of spines in the same neuron (Xu et al 2009a; Yang et al 2009). However,
relatively little is known about effects of exercise and learning on the morphology of
striatal MSNs.
The purpose of this study was to examine the effects of motor training on striatal
MSN morphology using the 6 weeks high-intensity treadmill exercise paradigm, the
Golgi-Cox technique and electron microscopy. The analysis focused on MSNs in the
dorsal striatum, the region that receives rich excitatory glutamate input from the motor
cortex and is involved in motor control. We examined dendritic spine density of MSNs in:
1) MPTP-lesioned, 2) control, 3) MPTP-lesioned plus exercise, and 4) control plus
exercise mice. Our results show significant increases in spine density on MSNs in both
MPTP and saline mice involved in treadmill exercise. Exercise differentially affected
different types of spines in MPTP-lesioned and control mice. In addition, electron
microscopic measurements showed increased number of synapses in response to
exercise in both MPTP-lesioned and control mice. There were no changes in the spine
area measurements using electron microscopy. Taken together, our results support the
97
hypothesis that enriched environment, learning and behavioral activity promote an
increase in spine density in the dorsal striatum.
4.3. Materials and methods
Mice selection and MPTP-lesioning
Male C57BL/6 mice 8 to 10 weeks old (Charles River Laboratories, Wilmington,
MA) and weighing between 25 and 30g were group-housed in a temperature-controlled
room under a 12h light/12h dark cycle with free access to water and standard rodent
food. All procedures were performed in accordance with the NIH Guide for the Care and
Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use
Committee at the University of Southern California. A total of 60 mice were used in this
study: (i) saline (n=13), (ii) saline plus exercise (n=17), (iii) MPTP (n=14), and (iv) MPTP
plus exercise (n=16). Only mice that were able to maintain forward position on a 6-lane
mouse treadmill (Model EXER-6M, Columbus, OH) for 5 min at the speed of 8m/min
were used for in experiments. For lesioning, 20 mice received 4 i.p. injections of
20mg/kg MPTP (free-base; Sigma-Aldrich, St. Louis, MO) in saline at 2h intervals or 4
injections of 0.1ml 0.9% NaCl as control. This lesioning regimen resulted in 68%
dopamine loss (77.9 ± 12.0 ng/mg of protein) in MPTP mice compared to saline mice
(246.9 ± 19.8 ng/mg of protein) measured by HPLC at the end of the study. Additionally,
there was 78% dopamine loss in MPTP plus exercise mice (69.8 ± 11.7 ng/mg of
protein) compared to saline plus exercise mice (315.2 ± 9.0 ng/mg of protein). Treadmill
exercise increased striatal dopamine levels (for 27%) only in saline plus exercise mice
compared to saline mice, and there was no significant change in striatal concentration
between MPTP and MPTP plus exercise mice. At the same time, there was a 60% loss
of synaptic expression of TH protein (rate limiting enzyme in dopamine synthesis) in the
98
dorsal striatum of both MPTP and MPTP plus exercise mice compared to saline and
saline plus exercise mice (F
(1,28)
= 138.3; p<0.05) and no significant effect of exercise
between the groups. Also, MPTP-lesioning produced 60-70% loss of substantia nigra
pars compacta (SNpc) neurons based on unbiased stereological counting (Jakowec et al
2004a; Petzinger et al 2007).
Treadmill exercise
Exercise was initiated 5 days after saline or MPTP injections. Mice from the two
exercise groups (saline plus exercise and MPTP plus exercise) were trained to run on a
100cm motorized treadmill belt (rodent treadmill, model Exer 6M, Columbus Instruments,
OH) at incremental speeds for 6 weeks (5 days /week) to reach the goal duration of 2 x
30 min/day, and goal speed of 20-22 m/min. A non-noxious stimulus (metal-beaded
curtain) was used as a tactile incentive to prevent mice from drifting back on the
treadmill (Fisher et al 2004; Petzinger et al 2007). All mice from saline plus exercise and
MPTP plus exercise were run together, and at the same time during the day.
Tissue collection for HPLC analysis of dopamine and its metabolites
Brain tissue from all mice was collected at the end of the study (corresponding to
47 days post-MPTP-lesioning). Brains were quickly removed and dorsal striatum was
dissected fresh en block corresponding to anatomical regions from bregma 1.2 to
bregma 0.6, with borders ventral to the corpus callosum, 2mm dorsal from the anterior
commissure and nucleus accumbens, medial to the external capsule, lateral to the
lateral ventricle according to the stereotaxic atlas of the mouse brain (Paxinos 2001).
Neurotransmitter concentrations of dopamine in striatal homogenates (n=4 per group)
were determined by HPLC with electrochemical detection as previously described (Irwin
99
et al 1992; Kilpatrick et al 1986; Petzinger et al 2007). The system consisted of an ESA
auto-sampler (ESA Inc., Chelmsford, MA) equipped with a 150 x 3.2 mm reverse phase
C-18 column (3µm diameter) regulated at 28°C and a CoulArray 5600A (ESA Inc,
Chelmsford, MA), equipped with a 4-channel analytical cell with potentials set at -
100mV, -30mV, 220mV and 350mV.
Morphometrical analysis of medium spiny neurons in dorsal striatum using Golgi-Cox
impregnation
Twenty four hours following the last exercise session, all mice were deeply
anesthetized with overdose of sodium pentobarbital (5 mg/kg) and perfused
transcardially with 100ml of 0.9% saline solution. Following removal, each brain was
processed using FD Rapid GolgiStain™ Kit according to the manufacturer’s protocol (FD
NeuroTechnologies, Ellicott City, MD). Briefly, each brain was incubated for 2 weeks at
room temperature in the impregnation solution containing potassium dichromate and
mercuric chloride (minimum 5mL per 1cm
3
of brain tissue). Following impregnation,
brains were transferred into solution containing sucrose for 48h at 4°C. Coronal 200µm
tick sections were cut on a Vibratome (Ted Pella Inc.) and mounted on 2% gelatin-
coated microscope slides. Approximately 15-17 sections spanning the entire striatum
were collected from each brain. Impregnation was visualized in developing solutions
provided with the Kit. Developed sections were rinsed in water, dehydrated in increased
concentrations of ethanol, cleared in xylene and coversliped in mounting medium
(Permount). Brain sections were examined and analyzed using Olympus BX-51
microscope equipped with motorized stage, a 60x water-immersion objective (Olympus
Inc.) and CCD camera (M12/Retiga 1300, QImaging, Canada). Medium spiny neurons
located in the dorsal striatum were digitally traced in x-, y-, and z-coordinates using
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Bioquant software package with neuron reconstruction algorithm (Bioquant Imaging,
Nashville, TN). Neurons whose dendrites can be traced within a single 200µm section
and not truncated were selected for tracing and analysis. Primary dendrites were
merged with cell soma. The 3-dimensional coordinates of all visible spines located on a
neuron of interest were recorded as points. The total number of spines per each neuron
was automatically summed using the software. The spine density (number of spines per
10µm) was calculated by dividing the total number of spines for each neuron by the total
traced dendritic length. Additionally, spine density per dendritic branch order for each
analyzed MSN was calculated using the following formula: total spine number
associated with the specific branch order / total dendritic length of that branch order.
Electron microscopy
Electron microscopic was carried out on mice from the saline (n=6), saline plus
exercise (n=10), MPTP (n=7), and MPTP plus exercise (n=9). Anesthetized mice were
perfused transcardially with 6 ml of heparin (1000 U/ml) in HEPES buffer (pH 7.3)
followed by 50ml of 2.5% glutaraldehyde/0.5% paraformaldehyde in HEPES (pH 7.3)
containing 0.1% picric acid. The brain was removed and post-fixed overnight at 4
o
C.
Vibratome sections (200mm thick) were cut in the coronal plane through the striatum
and the dorsal hippocampus. A 2x2mm
2
piece of the dorsolateral striatum (site of the
major input of the corticostriatal pathway) was dissected, washed in HEPES buffer,
incubated at room temperature in the dark in aqueous 1% osmium tetroxide/1.5%
potassium ferricyanide, washed in de-ionzed water and en block stained with aqueous
0.5% uranyl acetate at room temperature for 30 minutes. The tissue was dehydrated,
embedded in Embed 812/Spurr's (EMS, Fort Washington, PA) and thin sections were
cut (80nm) on an ultramicrotome (Leica Ultracut), placed on 200 mesh nickel coated
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grids and stained with uranyl and lead salts. Photographs (10/animal) were taken
randomly (1 section/grid, one photograph per grid square) at a final magnification of
40,000X within the area of the neuropil (location of the greatest number of synapses) by
an individual blind to the particular experimental group and then captured on the
computer using an AMT (2Kx2K) digital camera (Danvers, MA). The area of the
dendritic spine that was associated with a nerve terminal making an asymmetrical
(excitatory) synaptic contact was determined using image pro plus imaging software
(Media Cybernetics, Inc., Tacoma, WA). Only those spines in which a presynaptic
terminal could be seen making a synaptic contact were analyzed. The mean area of the
dendritic spine was calculated for each animal and then a grand mean determined for
that particular experimental group.
Statistical analysis
The differences between experimental groups in total spine density were
analyzed using two-way analysis of variance (ANOVA) with treatment as between
subject factor (saline vs. MPTP), and exercise as within subject factor (no exercise vs.
exercise). The Bonferroni post hoc test was used to correct for multiple comparisons
when assessing significance of interest. Analysis was performed using Prism5 for
Windows (Graph Pad, San Diego, CA). Significance level was set to p<0.05.
4.4. Results
The Golgi-Cox impregnation technique was used to uniformly label cells
throughout the striatum to examine changes in the number and morphology of dendritic
spines (Fig. 4.1A). MSNs, selected for the morphological analysis from the dorsolateral
striatum were differentiated from interneurons and glia by their unique cellular
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morphological characteristics, including a cell body diameter of about 15 to 18 µm, spiny
dendritic trees, and the presence of a single axon without spines (Chang et al 1982)
(Fig. 4.1B). Treadmill exercise significantly increased the average spine density in both
exercise groups compared to sedentary mice (F
(1,47)
= 57.01, p < 0.05) (Fig. 4.1C).
Average spine number per 10 µm dendritic length was 6.60 ± 0.21 for saline, 8.33 ± 0.23
for saline plus exercise, 5.03 ± 0.19 for MPTP, and 7.73 ± 0.44 for MPTP plus exercise
mice (Fig. 4.1D). The MPTP lesion significantly decreased average spine density (F
(1,47)
= 13.86, p < 0.05). There was no significant interaction effect of MPTP and exercise on
spine density (F
(1,47)
= 2.73, p > 0.05).
We further investigated if the shape of spines influenced differences observed in
total spine density. The average density of mushroom spines (per 10 µm of dendritic
length) was 0.72 ± 0.15 in saline, 0.62 ± 0.09 in saline plus exercise, 0.34 ± 0.08 in
MPTP and 0.95 ± 0.09 in MPTP plus exercise mice (Fig. 4.1E). Statistical analysis of
mushroom spine density showed a strong interaction effect of exercise and the MPTP
lesion (F
(1,47)
= 16.44, p < 0.05), indicating that the effect of treadmill exercise is larger in
lesioned mice compared to controls. Finally, we separately analyzed the density of all
non-mushroom spines and found that the number of spines per 10 µm dendritic length
was 5.83 ± 0.21 for saline, 7.71 ± 0.23 for saline plus exercise, 4.69 ± 0.19 for MPTP,
and 6.76 ± 0.44 for MPTP plus exercise mice (Fig. 4.1F). Exercise significantly
increased the average spine density in both groups when compared to sedentary mice
(F
(1,47)
= 40.95, p < 0.05). This effect was most pronounced on the fourth order dendrites,
while there was no exercise effect the second and third order dendritic spine density.
Additionally, there was a significant MPTP lesion effect on the density of all non-
mushroom spines (F
(1,47)
= 11.46, p < 0.05).
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Figure 4.1: High intensity treadmill exercise increases spine density on striatal MSNs.
(A) Representative coronal section of Golgi-Cox impregnated mouse brain (Bregma:
0.14mm) with delineated area of dorsolateral striatum (white dashed line) within which
MSNs were selected for analysis. Scale bar: 1mm. (B) Representative Golgi-Cox labeled
MSNs from dorsal striatum selected for the morphological analysis. Scale bar: 20µm; cc-
Corpus callosum. (C) Photomicrographs of MSNs dendritic branches from representative
mice from each of four experimental groups illustrate changes in spine density after
MPTP-treatment and in response to exercise. Scale bar: 10µm. (D) Bar graph illustrating
quantitative measurements of total dendritic spine density on MSNs in dorsolateral
striatum in saline (n=7 neurons), saline plus exercise (n=25 neurons), MPTP (n=9
neurons) and MPTP plus exercise (n=10 neurons). Treadmill exercise significantly
increased the average spine density in both exercise groups compared to sedentary
mice. Bar graphs illustrating quantitative measurements of mushroom (E) and non-
mushroom (F) spine densities on MSNs in dorsolateral striatum. Data are presented as
mean ± SEM. The symbols “#”, “&” and “*” represent significant effect of exercise, MPTP
lesion and MPTP x exercise interaction at p<0.05, two-way ANOVA.
In contrast to the spine density results, ultra-structural analysis of spine area
using electron microscopy (Fig. 4.2A) showed no significant effect of MPTP or exercise
(Fig. 4.2B). However, non-stereological counting of synapse number from all EM images
taken in a blinded fashion showed a small but significant increase in synapse number in
response to exercise in both treatment groups (F
(1,28)
= 4.21, p < 0.05). Average
synapse number in a 32 µm
2
field of view per group was: 2.85 ± 0.23 for saline, 3.16 ±
0.11 for saline plus exercise, 2.53 ± 0.21 for MPTP, and 2.93 ± 0.17 for MPTP plus
exercise mice (Fig. 4.2C).
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Figure 4.2: Electron microscopic analysis of spine area and number of synapses in
dorsal striatum of mice. (A) Electron photomicrograph from a control animal showing a
nerve terminal (NT) making an asymmetrical synaptic contact (arrowheads) with an
underlying dendritic spine (DS). The two points of the synaptic contact contain a
postsynaptic density that is perforated. The 10nm gold particles located inside the
terminal and in the dendritic spine show the location of an antibody against glutamate.
Scale bar: 100nm (B) Bar graph illustrating quantitative measurements of spine area,
and (C) non-stereological counting of the number of synapses per 32µm
2
field of view, in
dorsolateral striatum in saline (n=6), saline plus exercise (n=10), MPTP (n=7), and
MPTP plus exercise (n=9) treated groups. While there was no significant effect of MPTP
or exercise on spine area (B), exercise significantly increased the number of synapses
(C) in both treatment groups. Data are presented as mean ± SEM. The symbol “#”
represents a significant effect of exercise at p<0.05, two-way ANOVA.
105
4.5. Discussion
In the current study, we investigated the effects of high intensity treadmill
exercise on MSNs spine density, spine area, and number of synapses in the MPTP
mouse model of dopamine depletion. The results show a significant increase in dendritic
spine density on MSNs in both lesioned and control exercise groups. Similarly to
previous reports, MPTP lesion alone significantly reduced overall spine density on
MSNs. In MPTP-lesioned mice exposed to treadmill exercise dendritic spine density
increase resulted in normalization of MSNs spine density from the lesion state. In
addition, analysis of Golgi stained MSNs in dorsolateral striatum indicates differences in
types of spines that return in lesioned mice in response to treadmill exercise. Also, our
exercise paradigm also promoted synapse formation in the dorsolateral striatum in both
lesioned and control mice. There were no changes in spine area when analyzed by
electron microscopy.
Previous studies showed that learning is an important aspect of behavioral
activity necessary to produce lasting morphological correlates of synaptic plasticity.
Challenging acrobatic training in healthy rats results in more synapses on Purkinje cells
in the cerebellum when compared to exercised or inactive animals (Black et al 1990),
suggesting that motor learning during acrobatic training and not repetitive neuronal
activation is required for new synapse formation in the cerebellum. Similarly, learning
new motor skills is associated with formation of new spines in cortical pyramidal
neurons, followed by loss of a different set of spines in the same neuron (Xu et al 2009a;
Yang et al 2009). Interestingly, this process was also associated with the loss of a
different set of spines, resulting in the maintenance of a constant total number of spines
in the motor cortex.
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Exercise repetitively activates circuits within the basal ganglia and this pattern of
activation can lead to long-term synaptic plasticity. To test this hypothesis, we used the
MPTP mouse model and treadmill exercise to investigate morphological correlates of
synaptic plasticity on MSNs in the dorsolateral striatum. Observed spine density
increase on MSNs could indicate enhanced activity of the motor cortical networks
projecting to the dorsal striatum. In vitro studies of cultured MSNs with and without
cortical excitatory afferents strongly support this interpretation (Kossel et al 1997; Segal
et al 2003). Activation of the corticostriatal pathway results in an increase in the number
of synapses containing a perforated postsynaptic density (Meshul et al 1996). Many of
these perforated postsynaptic densities are associated with mushroom spines. Also,
formation of new spines and potentially new synapses, as we observed in our study,
could be a mechanism by which parallel or alternative connections are made between
the neurons in the striatum that belong to the same network. Studies of striatal MSNs
morphology in rats reared in enriched environments (Comery et al 1996) support for this
interpretation. Enriched environment significantly increases the density of split or
multiple headed dendritic spines on striatal MSNs when compared to rats reared in
single cages. It was suggested that formation of multiple headed spines on MSNs
reflects the strengthening of existing connections (via formation of parallel synapses), or
alteration of connectivity patterns (via addition of novel synaptic inputs).
Significant spine loss in the striatum following dopamine depletion appears to be
a key morphological event leading to functional changes in corticostriatal
neurotransmission in the parkinsonian state (Day et al 2006; Deutch et al 2007; Smith et
al 2009b)(Narushima et al 2006). On the other hand, previous studies using the MPTP-
lesioned mouse have shown that high intensity treadmill exercise modulates dopamine
neurotransmission through increased evoked dopamine release and elevated dopamine
107
D2 receptor expression (Petzinger et al 2007), as well as glutamatergic
neurotransmission, evident by elevated expression of the GluR2 AMPA receptor subunit
and altered MSNs excitability (VanLeeuwen et al 2010). Effects of high intensity treadmill
exercise on striatal circuits could be further investigated using the BAC-EGFP transgenic
mouse line that delineates between direct and indirect pathway MSNs in the striatum
(Gong et al 2003). Morphological analysis in combination with dopamine receptor D1
and D2 immuno-gold labeling at the ultrastructural level would give valuable insight as to
whether the affect of exercise on synapse formation seen in the present study is
selectively restricted on one of the striatal MSNs pathways.
4.6. Conclusions
In the present study we reported exercise-induced increases in spine density on
striatal MSNs and synapse number increases in dorsal striatum in the MPTP mouse
model of PD. To the best of our knowledge, this is the first report of exercise-induced
changes in neuronal morphology in an animal model of a neurodegenerative disease.
Our observation that high intensity treadmill exercise can reverse spine loss in the dorsal
striatum suggests new possibilities for symptomatic treatment of PD and other
neurodegenerative diseases and opens new possibilities for using activity-dependent
synaptic plasticity mechanisms in treatments of brain disorders.
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CHAPTER 5:
CHANGES IN AMPA RECEPTOR EXPRESSION AND MEDIUM SPINY NEURONS
MORPHOLOGY WITH TREADMILL EXERCISE IN THE 1-METHYL -4-PHENYL-
1,2,3,6-TETRAHYDROPYRIDINE-LESIONED MOUSE MODEL OF BASAL GANGLIA
INJURY
4
5.1. Abstract
Glutamate neurotransmission from the motor cortex to the dorsal striatum is an
important modulator of the basal ganglia activity. Medium spiny neurons (MSNs) in the
dorsal striatum are targets for glutamate afferents from the motor cortex and the
thalamus and dopamine input from substantia nigra pars compacta (SNpc). These two
inputs converge onto the spines of MSNs and modulate their activity. Due to severe
dopamine loss, patients with Parkinson’s disease (PD) and animal models of basal
ganglia injury develop aberrantly high corticostriatal glutamate excitability, suggested to
be a pathological adaptation to neuronal injury. Behavioral activity, such as daily
treadmill exercise, ameliorates MSNs excitability by mechanisms of reduced
conductance and changes in the subunit composition of glutamate alpha-amino-3-
hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. However, evidence in the
literature suggests that the direct and indirect striatal pathways are not equally sensitive
to dopamine loss, and the question remains if these two pathways share the same
molecular mechanism for experience-dependent synaptic plasticity. The present study
investigates AMPA receptor expression and conductance in the direct and indirect
pathway MSNs in the dorsal striatum of MPTP lesioned mice after 6 weeks of treadmill
exercise. To delineate between the two striatal pathways, we used BAC Drd2-eGFP
4
This chapter is in preparation for publication in the Journal of Neuroscience.
109
mouse transgenic line with eGFP expression selective to dopamine receptor D2
containing MSNs of the indirect pathway. Our results indicate that high intensity treadmill
exercise: (a) increases spine density of indirect pathway MSNs, (b) increases GluR2
AMPA receptor subunit expression in indirect pathway MSNs, and (c) promotes AMPA
GluR2 subunits insertion into the postsynaptic density of MSNs in the dorsal striatum.
These studies suggest that modulation of AMPA receptor expression and subunit
composition in indirect pathway MSNs provide potential mechanisms underlying activity-
dependent neuroplasticity of the injured basal ganglia.
Keywords: dopamine, glutamate, striatum, spine density, electrophysiology, MPTP,
animal models
5.2. Introduction
Experience-dependent brain plasticity is an important process for the function of
an adult brain. Research with animal models provides evidence that enriched
environment and learning of a new motor skill induces lasting synaptic plasticity of
glutamate neurotransmission in multiple brain regions, including the cerebral cortex,
hippocampus, and cerebellum (Black et al 1990; Dietrich et al 2005; Kleim et al 1996;
Naka et al 2005; Vasuta et al 2007; Xu et al 2009a; Yang et al 2009). Recent studies
show that physical exercise induces neuroplasticity in the basal ganglia via mechanisms
of glutamate neurotransmission modulation in the dorsal striatum (VanLeeuwen et al
2010). Given the complex anatomy and circuitry within the basal ganglia, and the ability
of dopamine to critically influence their activity through existence of the direct and
indirect striatal pathways, (each one having the opposing effect on motor output), the
110
next question to be answered is whether the two pathways share the same molecular
mechanisms of experience-dependent synaptic plasticity.
Excitatory glutamate neurotransmission is an important modulator of basal
ganglia activity, and thus influences motor function. Cortical glutamatergic inputs enter
the basal ganglia through the striatum, where medium spiny neurons (MSNs) represent
the majority of the neuronal population. Excitatory cortical afferent fibers make active
synapses on heads of dendritic spines and shafts of MSNs (Calabresi et al 1993;
Cepeda et al 2001b; Wang & Pickel 2002). Striatal MSNs also receive dopaminergic
input from midbrain areas to the neck of dendritic spines (Ariano et al 1997; Pickel et al
1981). The length of an average MSN dendritic spine is about 1-2 microns, and
dopamine and glutamate-containing axonal terminals converging on the same neuron
are thought to be in very close proximity thus influencing each other’s neurotransmission
(Smith & Bolam 1990). Activation of motor cortex circuitry in response to behavioral
activity releases glutamate to the dorsal striatum, and influences excitability of its MSNs
targets. Electrophysiological recordings from rat dorsal striatum showed that dopamine
attenuates all excitatory responses within MSNs, which is dependent on AMPA
receptors, and at the same time strongly potentiates NMDA receptor-dependent activity.
This selective gating of glutamatergic responses is dependent of dopamine receptor
profiles within the striatum: expression and activation of dopamine receptor D1 receptor
subtype promotes excitatory inputs, whereas inhibition depends on both, D1 and D2
receptor subunits (Cepeda et al 1993).
Within the basal ganglia, the alpha-amino-3-hydroxy-5-methyl-4-
isoazoleproprionic acid (AMPA) receptor subtype is responsible for the majority of fast
excitatory glutamate neurotransmission (Calabresi et al 1997b; David et al 2005; Starr
1995a). The AMPA receptors often co-localize with N-methyl-D-aspartate (NMDA)
111
receptors and voltage-gated calcium channels on post-synaptic membranes and play an
important role in modulating the electrophysiological properties of neurons, including
striatal MSNs. Functional AMPA receptor exists as a tetramer consisting of subunits
GluR1 through GluR4. The most abundant subunits within the striatum are GluR1 and
GluR2. Subunit composition and phosphorylation states of AMPA receptors regulate
synaptic connectivity and strength, including long-term potentiation (LTP) and long-term
depression (LTD). The GluR2 subunit plays a critical role in AMPA receptor activity as
its insertion into the membrane leads to formation of channels with decreased current
conductance, low Ca
2+
permeability, and outward rectifying current. Alternatively,
phosphorylation of GluR2 subunit at Ser880 by protein kinase C, leads to receptor
internalization and recycling from the synaptic membrane. Together, GluR2 up-
regulation and phosphorylation are two molecular events leading to the reduction of
glutamate synaptic strength.
Functional consequence of loss of striatal dopamine is increased excitability of
MSNs (Arbuthnott et al 1998; Betarbet et al 2000; Calabresi et al 2007; Chen et al
2001b; Liang et al 2008; Mallet et al 2006; Raju et al 2008; Starr 1995a). Elevated
excitability is caused by increased glutamate neurotransmission and loss of dopamine
receptors on axon terminals of striatal afferent cortical fibers (Calabresi et al 1993). In
vitro recordings from MSNs showed increased amplitude and frequency of spontaneous
excitatory postsynaptic currents (sEPSCs) in dopamine-depleted animals (Calabresi et
al 1993; Cormier & Kelly 1996; Tseng et al 2001). Application of an AMPA receptor
antagonist on slice preparations reversibly blocks spontaneous depolarizing
postsynaptic potentials, suggesting that modulation of this type of glutamate receptor is
responsible for elevated excitability of striatal neurons.
112
Daily treadmill running ameliorates this hyper-excitability of striatal MSNs, but
molecular mechanisms underlying this process are not well understood. Potential
molecular candidates involved in this phenomenon are trafficking and subunit
composition of AMPA glutamate receptors and dopamine receptor D2. A recent study
proposed this mechanism in experience-dependent plasticity of the basal ganglia in both
healthy and injured states (VanLeeuwen et al 2010).
Recent studies have shown that motor skill learning causes long-term changes in
potentiation and glutamate neurotransmission on striatal MSNs, and that this
phenomenon is region and task specific, meaning that with the extensive training
neurons within the dorsolateral striatum show significant change in their potentiation and
that this is selectively happening on striatonigral (indirect pathway) MSNs.
In the present study, we used BAC-Drd2-eGFP transgenic mice to delineate between
direct and indirect striatal MSNs. In these mice, enhanced Green Fluorescent Protein
(eGFP) is expressed under the promotor for dopamine D2 receptor. As a consequence,
MSNs involved in the indirect pathway of the basal ganglia circuits are endogenously
labeled with eGFP. Our findings suggest that high intensity treadmill exercise: (a) up-
regulates AMPA GluR2 receptor subunit within direct and indirect basal ganglia pathway,
(b) causes an increase of GluR2 subunit insertion into the postsynaptic density of MSNs,
and (c) causes a long-lasting up-regulation of GluR2 and altered dendritic and spine
morphology in indirect pathway MSNs.
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5.3. Materials and methods
Maintaining transgenic mouse colony and genotyping
All experiments were carried out in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals and approved by the
University of Southern California Institutional Animal Care and Use Committee (IACUC).
Transgenic mice used for this study Tg(Drd2-EGFP)118Gsat/Mmnc had enhanced
green fluorescent protein (eGFP) expression under the control of dopamine receptor D2
in MSNs of the indirect pathway (Gong et al 2003; Kreitzer & Malenka 2007). The
original background of mice was Swiss Webster and they have been crossed with
C57Bl/6 strain for 6 generations. Mice were housed in a temperature-controlled room
under a 12-hour shift in light/dark cycle. Adult (2-6 months old) GFP positive males and
females were used for breeding and maintenance of the colony.
Mice selection and MPTP-lesioning
Male 8-12 weeks old and eGFP positive mice were used for experiments. A total
of 72 mice were used: 18 mice per experimental group (i) saline, (ii) saline plus exercise,
(iii) MPTP, and (iv) MPTP plus exercise. Mice were handled daily prior to neurotoxin
administration. MPTP (Sigma, Inc, St. Louis, MO) was administered in a series of 4 i.p.
injections of 20 mg/kg (free-base) at 2-hour intervals. This regimen leads to
approximately 60% loss of nigrostriatal neurons and an 80-90% depletion of striatal
dopamine levels (Jackson-Lewis et al 1995; Jakowec et al 2004a; Przedborski et al
1995). Control mice received 4 i.p. injections of 0.9% NaCl.
114
Treadmill exercise protocol
Two weeks before the start of treadmill running, mice were acclimated to a 6-lane
mouse treadmill (Model EXER-6M, Columbus, OH) by placing them into the non-moving
treadmill for 5min. Then, the treadmill was turned on lowest speed (6m/min). Mice that
were able to maintain the forward position on the treadmill for 10min at 6m/min speed
were used for experiments. Daily treadmill running started 5 days after the MPTP-
lesioning or saline injections. Previous work demonstrated that by this time point MPTP-
induced neuronal death is complete (Jakowec et al 2004a). Running duration was
incrementally increased starting with 30min to reach the goal duration of 2x30min, with a
5min warm-up period at the beginning. Mice run 5 days/week for 6 weeks (Fisher et al
2004; Petzinger et al 2007). A metal beaded curtain and shock-plates were used as
tactile incentives to prevent animals from drifting back on the treadmill. Treadmill speed
for each group was increased gradually every 10min. Warm-up speed was 8m/min and
the target maximum speed was 22m/min. Non-exercised mice were be handled each
day and placed in the non-moving treadmill for the same amount of time as exercised
mice.
Tissue collection
Brain tissue from all groups of mice was collected on the last day of exercise,
corresponding to 47 days post-MPTP lesion. For fresh tissue analysis, mice were killed
by cervical dislocation (for HPLC, electrophysiology and synaptoneurosome
preparations). Brains were quickly removed and regions of interest identified using a
standard mouse brain atlas (Paxinos 2001). For HPLC and synaptoneurosome
preparations, dorsal striatum was dissected fresh en block corresponding to anatomical
regions from bregma 1.20 to 0.60, with borders ventral to the corpus callosum, 2mm
115
dorsal from the anterior commissure, medial to the lateral ventricle, and lateral 2.50mm
form the midline. For immunohistochemistry, mice were killed by pentobarbital overdose,
followed by transcardial perfusion with fixative. Immunohistochemistry and
electrophysiology were preformed on coronal sections corresponding to bregma 1.30 to
0.10.
HPLC analysis of striatal dopamine and its metabolites
Dopamine concentrations were determined by HPLC with electrochemical
detection as previously described (Irwin et al 1992; Kilpatrick et al 1986; Petzinger et al
2007; Vuckovic et al 2008). For this analysis, saline mice were killed at 7 days (n=4) and
45 days (n=3), and MPTP mice were killed at 7 days (n=6) and 45 days (n=3) following
the treatment. The system consisted of an ESA auto-sampler (ESA Inc., Chelmsford,
MA) equipped with a 150 x 3.2mm reverse phase C-18 column (3µm diameter) regulated
at 28°C and a CoulArray 5600A (ESA Inc, Chelmsford, MA), equipped with a 4-channel
analytical cell with potentials set at -100mV, -30mV, 220mV and 350mV. The HPLC was
integrated with a Dell GX-280 computer with CoulArray analytical program for Windows
(ESA Inc, Chelmsford, MA). Mobile phase consisted of acetonitrile in phosphate buffer
and an ion-pairing agent and was delivered at 0.6ml/min rate. Fresh frozen tissue was
homogenized in 0.4M HClO
4
, and centrifuged to separate precipitated protein. The pellet
was resuspended in 0.5M NaOH and used to determine total protein concentration with
the CoomassiePlus protein assay (Pierce, Rockford, IL) and microplate reader ELx800
(BioTek Instruments Inc., Winooski, VT) equipped with KCjunior software.
116
Immunochistochemistry analysis
For immunohistochemistry, a subset of mice (n=3 per group), were killed by
pentobarbital overdose (50mg/kg, i.p.) and transcardially perfused with 0.9% NaCl
followed by 4% paraformaldehyde in 0.1M phosphate buffered saline, pH 7.4 (4%
PFA/PBS). Brains were promptly removed and post-fixed in the perfusion fixative for 48h
at 4°C and cryopreserved in 20% sucrose in 0.1M phosphate buffer. Tissue was quickly
frozen in 2-methyl butane on dry ice and stored at -80°C. Coronal sections, 20µm-thick
through striatum (corresponding to Bregma 1.30 to 0.10), were cut using Leica 1900
cryostat (Leica Microsystems GmbH, Hussloch, Germany). After washing in 0.1M PBS
and blocking in 4% normal goat serum for 1h, tissue was exposed to 1:500 dilution of
anti-GluR2 monoclonal antibody (Antibodies Inc., Davis CA) for 48h at 4ºC. Sections
were washed and incubated in biotinylated goat anti-mouse secondary antibody (1:500;
Vector Laboratories, Burlingame, CA) for 18h at 4ºC. Staining was visualized by
exposure to avidin-Cy3.5 conjugate (1:500; Rockland Immunochemicals, Gilbertsville,
PA). Following extensive washes, sections were incubated in Hoechst 33324 nuclear
dye (1:10,000; Sigma), mounted on gelatin-covered slides and cover-slipped with
immersion oil for fluorescence (Olympus, Japan). Including controls where primary or
secondary antibodies were omitted from the protocol validated specificity of antibody
staining.
Quantification of GluR2 immunoreactivity in tissue slices
Relative expression of GluR2 protein was determined in striatal tissue sections
using a semi-quantitative analysis (VanLeeuwen et al 2010). Immuno-stained sections
were quantified for the relative expression of the intensity of GluR2 staining focusing on
(i) the cell bodies of dopamine receptor D2 expression MSNs (eGFP positive cells), and
117
(ii) the cell bodies of dopamine receptor D1 expressing MSNs (eGFP negative cells), in
the dorsolateral region of the mid-striatum (Bregma 0.74 to 0.38 mm). All analysis was
performed on an Olympus BX-DSU fluorescent microscope (Olympus, Japan) equipped
with a mercury fluorescent lamp, 100x oil objective, and interfaced with a Hammamatsu
CCD camera. Digital images of labeled neurons were captured in red, blue and green
channels separately. Images were analyzed off-line, using a computer assisted image
analysis program Methamorph (Molecular Devices, Downingtown, PA). For each data
image, a threshold based on fluorescence intensity in the red channel, was created in
order to automatically differentiate cell bodies from background or other artifacts. Using
the intensity established by a threshold tool in Methamorph software, MSNs cell bodies
were manually selected based on size (surface area between 250 and 300µm
2
),
morphology (appearance of dendritic arbor, large soma, and intact nucleus visualized in
the blue channel using a nuclear marker Hoechst 3324), location (within the perimeter of
the dorsolateral striatum). The relative signal intensity was automatically measured
within each cell body. Discrimination between dopamine D2 receptor expressing and
non-expression neurons was made by identifying each cell body in the green channel
image, using eGFP fluorescence expression. For each treatment group (saline, saline
plus exercise, MPTP, and MPTP plus exercise), sections from 6 mice were used for
analysis. Background corrections were calculated by subtracting the fluorescence
intensity from an oval region of interest (with an area of 250µm
2
) placed over the corpus
callosum from the same tissue section. Multiple sections form each of the four treatment
groups were processed in the same staining solutions and handled at the same time to
ensure that differences in immunoreactivity and signal intensity were due to antigen
expression. For each animal, in each treatment group, the average fluorescence
118
intensity of antibody-stained cells in the red channel was calculated for analysis. For
analysis all treatment groups were normalized against saline.
Electrophysiological studies
Mice from all groups (n=6 per group) were anesthetized in a dessicator
containing halothane vapors, killed by decapitation, and brains removed. Tissue was
blocked in ice-cold low-sodium sucrose-substituted saline (90mM NaCl with 105mM
sucrose) and striatal coronal sections were cut at 350µm thickness using a Vibratome-
1000 (Vibratome Co, St. Louis, MO). Slices were incubated in artificial cerebral spinal
fluid (aCSF consisting of 124mM NaCl, 1.3mM MgSO
4
, 3mM KCl, 1.25mM NaH
2
PO
4
,
25mM NaHCO
3
, 2.4mM CaCl
2
, and 10mM glucose) at room temperature (23ºC) for at
least one hour prior to recording. All solutions were continuously oxygenated with 95%
O
2
, 5% CO
2
. Tissue was transferred to a submerged brain slice-recording chamber
perfused with oxygenated aCSF kept at a recording temperature of 32ºC, as outlined by
Akopian and Walsh, 2007. The pH of all oxygenated solutions was 7.4. All experiments
were performed with 30µM bicuculline methiodide (BIC-aCSF). Picrotoxin (50µM) was
used to block gamma-amino butyric acid-A (GABA
A
) receptor mediated inhibition in an
attempt to isolate excitatory synaptic events.
Whole cell voltage clamp and electrical stimulation methods were used to
examine corticostriatal synaptic input to reduce possible activation of postsynaptic
conductances, which can contribute to changes in synaptic strength under current clamp
conditions (Akopian and Walsh, 2002). The eGFP positive MSNs were visualized using
a fixed stage fluorescent microscope with 40x and 60x water immersion lenses (Zeiss
Axioscope, Germany). Patch electrodes were pulled by a Flaming-Brown P-87 pipette
puller (Sutter Instrument, Novato, CA) from the thin-wall borosilicate capillary glass
119
1.5mm o.d. (WPI, Sarasota, FL) and filled with fresh solution of 5% biocytin (dissolved in
0.05M Tris, pH 7.4). The electrodes had resistances ranging between 4 and 6 MΩ when
filled with pipette solution. The pipette (internal) solution was composed of 130mM Cs-
gluconate, 10mM CsCl, 5mM EGTA, 10mM MgCl
2
2’ HEPES, 5mM QX-314, 2mM ATP-
Mg, and 0.25mM GTP-Na, pH 7.25, with 285mOsm. Spermine (100 µM; Sigma-Aldrich)
was included in some experiments to provide polyamine modulation of GluR2 lacking
AMPA receptors. The liquid junction potential between the pipette and aCSF was
estimated as 15mV. 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-msec hyperpolarizing (-5mV) pulses at the onset of
the traces 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 a 10mV depolarizing step voltage command from a
holding potential of 70mV.
Changes in the amplitude and frequency of spontaneous excitatory postsynaptic
currents (sEPSCs) were sampled for 2min. Neurons were voltage clamped at -70V
during recording. The membrane currents were filtered at 1kHz and digitized at 5kHz.
eEPSCs were analyzed off-line using the threshold detection option in Clampfit 9
(Molecular Devices, Sunnyvale, CA). The threshold amplitude for the detection of an
event was set above 5pA. Cumulative frequency histograms were generated for sEPSC
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amplitude and inter-event interval. The bin size for sEPSC inter-event interval was
100msec.
Stimulus-evoked excitatory postsynaptic currents (EPSCs) in the dorsal striatum
were generated using stimulation electrodes filled with aCSF and positioned 100-500µm
from the recording electrode, at the border between the striatum and the overlying
corpus callosum. Rectangular current pulses of 0.1msec duration were applied to the
stimulation electrode 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 software. Input (stimulation intensity) – output (synaptic response)
relationships were determined for corticostriatal synapses by applying a standard
ascending sequence of stimulus intensities and recording EPSCs. Neurons were voltage
clamped at -80mV during periods of stimulation. The slope of the input-output
relationship was determined for each cell and compared using two-way ANOVA. To
estimate the paired pulse ratio (PPR), five paired pulse synaptic stimulations with an
inter-stimulus interval of 50msec at holding potential of -70mV were delivered through
the stimulating electrode at 20sec intervals. The intensity of synaptic stimulation was set
at about 50% of maximum responses obtained from the I/O curve. All five traces were
averaged, and PPR was expressed as percentage of the ratio of the second pulse to the
first one in the series.
The rectification index (RI) was determined by the slope of the synaptic current-
voltage relationship (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). Synaptic current-voltage relationships (I-Vs) were obtained by
generating synaptic currents with electrical stimulation of cortical afferents every 20sec
at different holding potentials ranging from -80mV to +60mV, with increments of 20mV.
121
The stimuli were delivered 5sec after the stepped change in the holding potential. All RI
experiments were performed in slices bathed in picrotoxin to block GABA
A
receptor
mediated responses and AP-5 (50µM) to block NMDA receptor mediated responses
(VanLeeuwen et al 2010).
Morphological analysis of MSNs using biocytin labeling
MSNs from the indirect pathway were visualized using a fluorescent microscope
with 60x water immersion objective (Olympus, Japan). From each coronal hemi-section,
one D2-expressing MSN with a cell body located in the dorsal striatum and identified by
the green eGFP fluorescence was filled with biocytin. Following electrophysiological
recordings, sections were transferred into glass vials containing cold 4%
paraformaldehyde in 0.1M phosphate buffer with 0.9% NaCl, pH 7.4 and fixed for 24h at
4°C. Brain sections were then cryoprotected in 30% sucrose in 0.1M PB, quickly frozen
in 2-methyl-butane on dry ice and re-sectioned at 60µm using a cryostat (Leica,
Germany). Sections containing biocytin filled neurons were processed by incubation in
avidin-biotin-horseradish complex (ABC kit, Vector Labs, Burlingame) dissolved in 0.1M
Tris buffered saline with 0.05% Triton X-100at for 90min. The staining was visualized by
incubation in 0.1% w/w DAB and 0.1% v/v H
2
O
2
in 0.1M Tris buffer pH 7.4 for 30 min.
After washing, sections were mounted on gelatin-coated glass slides and dried
overnight. At the end, sections will be dehydrated in increasing concentrations of
ethanol (30%, 70% and 95%), cleared in xylene and coversliped with Permount.
Biocytin-filled MSNs were examined and analyzed using Olympus BX-51 microscope
equipped with motorized stage, a 60x water-immersion objective (Olympus Inc., Japan)
and CCD camera (M12/Retiga 1300, QImaging, Canada). MSNs were digitally traced in
x-, y-, and z-coordinates using Bioquant software package with neuron reconstruction
122
algorithm (Bioquant Imaging, Nashville, TN). Primary dendrites were merged with the
cell soma. The 3-dimensional coordinates of all visible spines located on a neuron were
recorded as points. The program automatically calculated the total number of spines per
neuron. During the analysis distinction was made between mushroom, thin and stubby
spines, based on their unique morphology. The lengths of all dendritic segments were
calculated from 3-dimensional coordinates in Excel (Microsoft). The spine density (in
number/µm) was calculated by dividing the total number of spines for each neuron by
the total dendritic length.
Statistical analysis
For all experiments, two-way analysis of variance (ANOVA) with exercise and
MPTP lesion as parameters was performed to compare the different groups and to
examine for significant interactions. A Bonferroni multiple comparison post-hoc test was
used to determine which groups are significantly different from others. Analysis was
performed using Prism5 for Windows (Graph Pad, San Diego, CA). Significance level
was set to p<0.05.
5.4. Results
MPTP administration significantly depletes striatal dopamine levels in Drd2-eGFP mice
To examine the effects of MPTP lesioning on striatal dopamine levels in Drd2-
eGFP transgenic mouse strain, we carried out HPLC analysis of dopamine in tissue
homogenates from the dorsal striatum. Dopamine levels were measured at 7 and 45
days post-MPTP lesioning (Fig 5.1). At 7 days after MPTP lesioning, there was a
significant loss of striatal dopamine in MPTP mice (40.95 ± 11.41ng dopamine/mg
protein) compared to saline treated mice (303.12 ± 20.61ng dopamine/mg protein). This
123
represents 87% dopamine depletion. At 45 days post MPTP, there was also significant
dopamine loss in MPTP mice (57.35 ± 13.46ng dopamine/mg protein) compared to
saline mice (320.77 ±17.83ng dopamine/mg protein) and this represents 82% dopamine
loss. At both time points, dopamine depletion was significant in MPTP mice compared to
saline mice (F
(1,21)
=192.4, p<0.01).
Figure 5.1: Dopamine levels in dorsal striatum of MPTP-lesioned and control BAC-Drd2-
eGFP mice. Seven and 45 days following MPTP or saline injections dopamine
concentrations were measured in tissue homogenates from the dorsal striatum in saline
(n=4 at 7 days and n=3 at 45 days), and MPTP (n=6 at 7 days and n=3 at 45 days) mice,
using HPLC. Data are presented as mean ± SEM. The symbol “*”
indicates statistically
significant difference between MPTP and saline treated mice at both time points
(p<0.05), two-way ANOVA.
Exercise Increases expression of AMPA GluR2 subunit in indirect pathway MSNs in both
saline and MPTP mice
Previous studies published from our lab using immunohistochemistry in MPTP
mice showed increased number of striatal MSNs expressing AMPA GluR2 receptor in
response to treadmill exercise (VanLeeuwen et al 2010). These studies did not delineate
between direct and indirect pathway MSNs. To investigate whether this effect is specific
to indirect pathway MSNs, we used BAC-Drd2-eGFP mice to investigate expression
levels of AMPA GluR2 receptor within eGFP positive MSNs in the dorsal striatum.
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Antibody specific for AMPA GluR2 was used for this experiment (Figure 5.2). Analysis
showed that exercise increased AMPA GluR2 receptor subunit in eGFP positive MSNs
specifically in MPTP mice in response to exercise (F
(1,28)
=8.225, p<0.05). There was also
a trend for MPTP effect on GluR2 expression (F
(1,28)
=3.954, p=0.056).
125
Figure 5.2: Analysis of GluR2 immunoreactivity in direct and indirect pathway MSNs
from striatal tissue sections of BAC-Drd2-eGFP mice, at the level of mid-striatum from
saline (n=3), saline plus exercise (n=2), MPTP (n=4), and MPTP plus exercise (n=3). (A)
Representative high magnification images (100x) of striatal tissue sections. Upper
panels: indirect pathway (eGFP positive) MSNs (in green). Middle panels: GluR2
immunoreactivity visualized using Cy3.5-conjugated antibody (in red). Lower panels:
merged images from green and red channels. Additional non-green neurons are visible
in red channel. Scale bar: 20µm. (B) Bar graph with relative fluorescent intensity in the
red channel (GluR2 immunoreactivity) measured in cell bodies of eGFP negative (direct
pathway) MSNs. (C) Bar graph showing relative fluorescent intensity in the red channel
(GluR2 immunoreactivity) measured in cell bodies of eGFP positive (indirect pathway)
MSNs. Data are presented as mean ± SEM. Exercise differentially regulated glutamate
neurotransmission by downregulating expression of GluR2 subunit in the control group
and significantly upregulating its expression in MPTP-lesioned mice in MSNs from both
direct and indirect striatal pathways. The symbol “*”
indicates statistically significant
interaction MPTP x exercise (p<0.05), two-way ANOVA.
126
127
Exercise increases thin spine density in indirect pathway MSNs in MPTP mice
Biocytin labeled indirect pathway MSNs in the dorsal striatum. These cells had
typical MSNs morphology: cell body diameter about 15-18µm, spiny dendritic tree, and
presence of a single axon without spines (Fig. 5.3A). Treadmill exercise significantly
increased average spine density in MPTP and saline mice compared to sedentary
controls (F
(1,18)
=7.65, p<0.05). Average spine number per 10µm dendritic length was
6.42 ± 1.17 for saline mice, 7.32 ± 0.47 for saline plus exercise, 5.52 ± 0.74 for MPTP,
and 6.69 ± 0.34 for MPTP plus exercise (Fig. 5.3B). MPTP lesion did not have a
significant effect on average spine density (F
(1,18)
=0.01, p>0.05). We further investigated
if the shape of spines influenced the differences observed in the total spine density. We
separately analyzed the density of thin, mushroom and stubby spine density on indirect
pathway MSNs (Fig 5.4). For mushroom spines, average density (per 10µm) was 1.42 ±
0.39 for saline mice, 1.13 ± 0.17 for saline plus exercise, 1.60 ± 0.27 for MPTP, and 1.23
± 0.21 for MPTP plus exercise (Fig. 5.3A). Statistical analysis showed no effect of
exercise (F
(1,18)
=0.00, p>0.05), or MPTP (F
(1,18)
=0.00, p>0.05) on mushroom spine
density. For thin spines, the number of spines per 10µm dendritic length was: 4.84 ±
0.66 for saline mice, 5.11 ± 0.33 for saline plus exercise, 4.27 ± 0.30 for MPTP mice,
and 5.75 ± 0.54 for MPTP plus exercise (Fig. 5.4B). There was a significant increase in
thin spine density in both exercise groups (F
(1,18)
=7.98, p<0.05). However, there was also
a strong interaction between exercise and MPTP lesion (F
(1,18)
=5.16, p<0.05), indicating
that the effect of treadmill exercise was larger in lesioned mice compared to control
mice. For stubby spines, average density (per 10µm) was 1.06±0.26 for saline mice,
1.08 ± 0.16 for saline plus exercise, 0.36 ± 0.05 for MPTP, and 0.25 ± 0.04 for MPTP
plus exercise (Fig. 5.3C). There was a significant MPTP effect on density of stubby
128
spines (F
(1,18)
=19.58, p<0.05), however, no significant exercise effect (F
(1,18)
=0.06,
p>0.05), and no interaction (F
(1,18)
=0.14, p>0.05).
Figure 5.3: High intensity treadmill exercise increases spine density on indirect pathway
striatal MSNs in MPTP-lesioned mice. (A) Upper panels show lower magnification (20x)
images of representative biocytin filled indirect pathway MSNs from each of the four
treatment groups. Scale bar: 100µm, cc-Corpus callosum. Lower panels show high
magnification (120x) images of dendritic segments with spines from representative cells.
Scale bar: 20µm. (B) Bar graph illustrating quantitative measurements of total dendritic
spine density on indirect pathway MSNs in saline (n=3 neurons), saline plus exercise
(n=8 neurons), MPTP (n=5 neurons) and MPTP plus exercise (n=6 neurons). (C) Bar
graph illustrating average spine density only on proximal dendrites. There was a
significant effect of exercise in both, MPTP-lesion group and the control. (D) Bar graph
showing average spine density on distal dendrites. Exercise increased spine density
only in MPTP-lesioned mice. Data are presented as mean ± SEM. The symbols “*” and
“#” represent significant MPTP x exercise interaction and significant effect of exercise,
respectively, at p<0.05, two-way ANOVA.
129
Figure 5.4: High intensity treadmill exercise differentially influences type of spines in
striatal MSNs in control and MPTP-lesioned mice. Upper panels: images of
representative (A) mushroom, (B) thin and (C) stubby spines at high magnification
(240x). Scale bar: 5µm. Lower panels: bar graph illustrating quantitative measurements
of mushroom, thin, and stubby spine density on indirect pathway MSNs. The effect of
exercise was significantly greater in MPTP-lesioned mice compared to saline controls for
thin spine density in (B). There was a significant effect of MPTP on stubby spine density
in (C). The symbols “*” and “&” represent significant effect of MPTP x exercise
interaction and MPTP lesion, respectively, at p<0.05, two-way ANOVA.
Exercise increases the rectification index for AMPA receptor-mediated synaptic
responses in indirect pathway MSNs in MPTP mice
To further investigate the effects of exercise on GluR2 subunit expression in
AMPA receptors in MPTP mice, we analyzed the rectification index in stimulus-evoked
current-voltage relationships from indirect pathway MSNs in the dorsal striatum. A
unique characteristic of AMPA receptor lacking GluR2 subunit is presence of inward
rectification in electrophysiological recordings (Bowie & Mayer 1995; Kamboj et al 1995).
This is due to Ca
2+
permeability of GluR2-lacking receptors and its sensitivity to cellular
polyamines. In contrast, AMPA receptors containing GluR2 subunits show linear current-
voltage relationship during recordings and insensitivity to polyamines (Pellegrini-
Giampietro et al 1997). In the present study, we stimulated MSNs from the dorsolateral
striatum by placing a stimulating electrode in the overlying corpus callosum. Evoked
synaptic responses were recorded at different holding potentials (from -80mV to +60mV,
130
with 20mV increments) by whole cell patch clamp method with a recording electrode
filled with spermine (100µM). Using these settings, a low rectification index (RI) is an
indication of inward rectification of AMPA receptor lacking GluR2 subunit, while high RI
indicates presence of AMPA receptors with GluR2 subunit. Recordings from eGFP
positive MSNs showed significant effect of exercise on RI in both saline plus exercise
(0.84 ± 0.02) and MPTP plus exercise (0.87 ±0.02) groups (F
(1,47)
=5.417, p<0.05).
However, the effect of exercise in RI was larger in MPTP plus exercise mice compared
to MPTP mice (0.77 ± 0.02) than in saline plus exercise compared to saline mice (0.86 ±
0.02) (Fig. 5.5A), since there was a significant interaction between MPTP x exercise
(F
(1,47)
=11.96, p<0.05). There was no significant effect of MPTP on RI (F
(1,47)
=3.201,
p=0.08). Taking together, these results support findings from immunohistochemical
analysis and indicate that exercise promotes expression of GluR2 containing AMPA
receptors in indirect pathway MSNs of MPTP mice.
Figure 5.5: Treadmill exercise reduces inward rectification of AMPA receptor mediated
excitatory postsynaptic currents on indirect pathway MSNs in MPTP mice. (A) Bar graph
showing the measurements of the rectification index (RI). Excitatory synapses on
indirect pathway MSNs demonstrate significantly lower RI compared to MPTP plus
exercise mice. (B) Bar graph showing AMPA/NMDA receptor ratio for indirect pathway
MSNs. Data are presented as mean ± SEM. The symbol “*” represents a significant
affect at p<0.05, two-way ANOVA.
131
Exercise does not affect eEPSC or the input-output measurements in indirect pathway
MSNs
Exercise-driven increase in GluR2 subunit expression in indirect pathway MSNs
could lead to a change in their excitability. We tested this hypothesis by recording
spontaneous excitatory post-synaptic currents (sEPSCs) from eGFP positive striatal
neurons (Figure 5.6). Our analysis included recordings of sEPSCs amplitude (which
reflects changes in the receptor number or ion channel conductance on the postsynaptic
side of the synapse), and recordings of sEPSCs frequency (whose changes reflect
modulations of presynaptic glutamate neurotransmission and its release) (Manabe et al
1993; Maren 1993). In this study electrophysiological recordings showed no significant
difference in amplitude or frequency on sEPSCs in indirect pathway MSNs. Also, we did
not observe any significant changes in measurements of paired pulse ratio (data not
shown). In addition, there were no significant differences in input/output relationship at
corticostriatal synapses in indirect pathway MSNs in any of the treatment groups (Figure
5.7).
Figure 5.6: Exercise does not influence amplitude or frequency of sEPSCs in indirect
pathway MSNs. Cumulative frequency histogram s of sEPSCs amplitude (A) and
frequency (B). Data are presented as mean ± SEM. No significant differences were
observed between the groups.
132
Figure 5.7: Exercise does not influence the input-output relationship for corticostriatal
synapses on indirect pathway MSNs. (A) Graph showing a relationship between stimulus
intensity (input) and EPSC amplitude (output) from corticostriatal synapses at indirect
pathway MSNs from the four experimental groups. (B) Bar graph showing calculated
slopes of the input-output relationship for corticostriatal synapses on indirect pathway
MSNs, as shown in A. Data are presented as mean ± SEM. There was no significant
effect of MPTP or exercise on the measurements of input-output relationships.
5.5. Discussion
Subunit composition of glutamate AMPA receptor and dopamine D2 receptor, are
critical for experience-dependent synaptic plasticity in the striatum. The present study
focuses on their interaction within the indirect pathway MSNs in the dorsal striatum, the
part of the basal ganglia most severely affected by dopamine depletion. Our
experimental approach utilized a transgenic mouse line with eGFP expression selective
to dopamine receptor D2 containing MSNs (the indirect pathway). In the present study,
we report that high intensity treadmill exercise leads to morphological correlates of
synaptic plasticity on indirect pathway MSNs, as measured by an overall increase in
spine density in exercised versus sedentary MPTP-lesioned mice. The observed spine
density increase was accompanied by modulation of glutamate neurotransmission
133
associated with changes in AMPA receptor expression and conductance in the indirect
pathway MSNs of the dorsal striatum.
The degree of dendritic spine loss on striatal MSNs greatly correlates with the
severity of dopamine depletion (Smith et al 2009b). However, in the present literature,
there is a controversy regarding the specificity of spine loss towards the indirect or direct
pathway MSNs. A study utilizing transgenic BAC D1 and D2 mouse lines and well as 6-
OHDA lesioned rats reported a selective loss of spines on indirect pathway MSNs (Day
et al 2006). However, electronmicroscpy analysis of spine density in MPTP-lesioned
monkeys revealed that MSNs from both pathways were affected (Smith et al 2009b;
Villalba et al 2009).
There is compelling evidence in the literature that the loss of midbrain dopamine-
producing neurons is responsible for increased cortical glutamate drive to the striatum,
specifically targeting MSNs, and contributing to the motor deficits in PD (Cepeda et al
2001a; Meshul et al 1999; Neely et al 2007; Schwarting & Huston 1996). A recent study
proposed decrease in AMPA receptor conductance on MSNs as a possible mechanism
for diminishing corticostriatal hyperactivity in response to treadmill exercise
(VanLeeuwen et al 2010). Furthermore, AMPA receptor antagonists alleviate motor
symptoms in animal models of PD (Chase & Oh 2000; Klockgether et al 1991).
Previous studies using the MPTP-lesioned mouse showed that high intensity
daily treadmill running leads to decreased glutamatergic excitability within the dorsal
striatum. This was evident on multiple levels on analysis: immuno-electron microscopy
reveled that treadmill exercise reversed the MPTP-induced increase in presynaptic
glutamate immunolabeling within striatal terminals (Fisher et al 2004). Previous studies
showed that treadmill exercise increases GluR2 subunit expression and phosphorylation
at Ser 880 in the dorsolateral striatum of MPTP-lesioned mice (VanLeeuwen et al 2010).
134
Furthermore, changes in AMPA receptor subunit expression were accompanied by a
reduction of excitatory postsynaptic currents in MSNs, suggesting that decreased MSNs
excitability can result from exercise-induced alterations in AMPA receptor subunit
expression.
135
CHAPTER 6:
CONCLUSIONS
Studies presented in this dissertation explored the molecular, cellular and
behavioral correlates of neuroplasticity in injured basal ganglia. Neuroplasticity is the
capacity of the nervous system to adapt and change its responsiveness following
repetitive activation events. Neuroplasticity encompasses a broad spectrum of chemical,
molecular, cellular, and physiological processes within the brain, from modulations in
neurotransmitter release to changes in neuronal architecture and network connectivity. A
fascinating aspect of neuroplasticity is that it can be initiated by pathological processes,
such as traumatic brain injury, or during the course of a neurodegenerative disease, but
also by environmental stimuli and behavioral activity. Studies in this dissertation examine
neuroplasticity in injured basal ganglia from two different aspects: during a
neurodegenerative process (in Chapter 2), and in response to behavioral activity,
specifically physical exercise in the form of high intensity treadmill exercise (in Chapters
3, 4 and 5). Chapter 2 focuses on relatively little investigated plasticity in the serotonin
system in response to the basal ganglia injury and severe dopamine loss throughout the
brain. Chapter 3 explores behavioral activity-dependent synaptic plasticity within the
basal ganglia and focuses on dopamine signaling and in particular on expression of
dopamine receptors. Chapter 4 further investigates activity-dependent synaptic plasticity
in the basal ganglia from the prospective of neuronal morphology and synapse
formation. Finally, Chapter 5 builds upon discoveries from preceding chapters and uses
modern tools of molecular biology and electrophysiology to examine detailed molecular
and cellular mechanisms of activity-dependent synaptic plasticity in the injured basal
ganglia focusing on glutamate neurotransmission.
136
6.1. Chapter 2: basal ganglia injury causes impairments in non-motor behavior
Chapter 2 focuses on little investigated changes in serotonin neurotransmission
throughout the brain in response to basal ganglia injury and severe dopamine depletion.
Studies in this chapter also investigate non-motor behavior, such as mood, memory and
fear conditioning, due to their sensitivity to changes in serotonin neurotransmission. It is
well-established that perturbation of serotonin neurotransmission in normal individuals
can lead to depression, anxiety, and memory impairment (Mann & Yates 1986; Mann
1999; Pillon et al 1989b). In addition, patients with PD develop low cortical serotonin
levels and degeneration of the dorsal raphe nucleus (Agid et al 1989; Cummings 1992;
Gotham et al 1986; McCance-Katz et al 1992; Scatton et al 1983). Studies in Chapter 2
used the MPTP neurotoxic lesioning in the substantia nigra pars compacta (SNpc) that
produces severe dopamine depletion in mice and non-human primates, and causes
significant decrease of serotonin across multiple brain regions. Results showed that
MPTP mice exhibited impairment in associative memory and fear conditioning at two
different time points following the lesion.
Despite serotonin depletion in brain regions important for affective behavior, our
MPTP-lesioning mouse did not show significant changes in anxiety and depression. The
lack of behavioral effect could be explained by (i) the level of serotonin depletion may
not be severe enough to manifest elevated anxiety, or (ii) the affected neurotransmitter
systems may compensate to overcome perturbation. The serotonin system following
MPTP-lesioning may compensate in an analogous fashion to that of the dopamine
system due to increased dopamine receptor expression (Betarbet & Greenamyre 2004).
Also, it is possible that the emergence of depression and anxiety in this mouse model
depends upon greater serotonin loss in critical brain regions.
137
A number of possible future studies could further examine the plasticity of
serotonin system in injured basal ganglia and its role in development of non-motor
behavioral impairments. Studies presented in Chapter 2 did not examine expression of
serotonin receptors or serotonin transporter (SERT) in the critical brain regions
(prefrontal cortex, amygdala and ventral striatum). Studies of serotonin receptors in
injured basal ganglia could add some insight to why we were not able to detect mood
disorders in the MPTP mouse. Serotonin receptors, and particularly 5-HT2 and 5-HT1
isoforms, are widely expressed in the basal ganglia where they display inhibitory and
excitatory function on the dopamine system (Di Matteo et al 2002; Fox & Brotchie 2000a;
b). Since studies in Chapter 2 found significant serotonin loss in the amygdala, frontal
cortex and striatum following MPTP lesion, a follow up study of expression levels of
serotonin receptors in these brain regions would contribute to our understanding of
interactions of these two neurotransmitter systems and their relation to the development
of behavioral symptoms. Studies of SERT in striatal and non-striatal brain regions
following dopamine depletion are of particular interest as inhibition of serotonin re-uptake
on nerve terminals significantly reduces symptoms of depression and anxiety in human
patients with PD (Chen 2004; Kostic et al 1987; Leentjens et al 2006; Mann 1999;
Mayeux 1990; Menza et al 2006). Additionally, compounds that inhibit SERT are widely
used in clinical practice to manage mood disorders in PD patients. Molecular
mechanisms that regulate expression levels and activity of SERT in dopamine-depleted
brain are not well understood, therefore animal models of basal ganglia injury, such as
the MPTP mouse, provide a good model system for analysis. In addition, using a mouse
model allows one to take advantage of genetic tools available in mice, such as
transgenic expression of mutated forms of SERT that occur in humans and use these
mice to study the serotonin system following basal ganglia injury.
138
Also, long-term effects of dopamine lesions to the serotonin system are still
poorly understood. For these studies, acute dosage of MPTP to mice may not be the
best model system, since in mice spontaneous return of striatal dopamine occurs within
3 to 6 months following the neurotoxin administration (Jakowec et al 2004b). A possible
alternative model would be the chronic administration of MPTP, with daily doses over a
long period of time (weeks to months). Studies of long lasting effects of MPTP
neurotoxicity in marmosets reported a marked and lasting serotonin depletion in the
striatum, nucleus accumbens, hypothalamus and cortical areas (Perez-Otano et al
1991a). It would be of great importance to study the effects of dopamine depletion in
aged animals, as PD affects predominantly older population in humans. Studies
presented in Chapter 2 used young adult mice age 8-10 weeks because acute MPTP
administration to aged mice causes high mortality due to the systemic effects of the
neurotoxin. For aged mice, chronic low daily dose of MPTP over a period of 3 months
could be an efficient way to produce severe dopamine depletion in the basal ganglia and
investigate serotonin levels in non-striatal brain regions. One can make a hypothesis that
the aged brain will have lowered capacity to compensate for dopamine loss and
serotonin levels would be severely depleted in the frontal cortex, amygdala and ventral
striatum, leading to changes in mood and memory performance.
6.2. Chapter 3: activity-dependent plasticity of dopamine neurotransmission
Chapter 3 reported exercise-induced plasticity of dopamine neurotransmission in
the basal ganglia, and in particular in the striatum. The dopamine D1 and D2 receptors
are the primary targets of dopamine on MSNs within the striatum and modulate their
physiological properties and cellular signaling. The dopamine D2 receptor is known to
play a major role in encoding motor skills within the dorsolateral striatum (Chen et al
139
2001a; Tinsley et al 2009). Studies in Chapter 3 showed that high intensity treadmill
exercise led to an increase in striatal dopamine D2 receptor expression and availability
for ligand binding; the effect was more pronounced in MPTP compared to saline treated
mice.
Studies in Chapter 3 showed for the first time exercise-induced plasticity of
dopamine D2 receptor in mouse brain using live imaging. To accomplish this, we used in
vivo positron emission tomography (PET) imaging and [
18
F]fallypride, a specific D2
receptor ligand, to measure receptor binding potential in the striatum at the end of 6
weeks of treadmill exercise. Furthermore, in order to provide support to PET measures,
studies in this chapter used the complementary technique of western immunoblotting to
measure changes in dopamine receptor protein expression in the same animals. The ex
vivo protein expression analysis showed significant increase in dopamine D2 receptor
expression in MPTP mice in response to exercise, and no change in dopamine D1
receptor expression between the experimental groups. Based on these results, it is
possible to hypothesize that the benefits of high intensity exercise are to enhance
dopamine signaling through increased dopamine D2 receptor expression in the striatal
indirect pathway, but not the dopamine D1 receptor of the direct pathway.
The enhanced effect of exercise in the MPTP-lesioned mouse may reflect an
attempt of the injured brain to optimize dopaminergic neurotransmission through
increased receptor number while dopamine levels remain low. Increased
responsiveness of MPTP mice to exercise indicates a greater potential of the injured
versus the healthy basal ganglia to undergo neuroplasticity leading to behavioral
recovery. Such a response in dopamine D2receptor expression may not be as essential
when striatal circuitry is intact.
140
PET imaging with dopamine receptor radiotracers offers the ability to carry out
longitudinal studies on the effect of exercise in animals and humans. Long term effects
of exercise on dopamine D2 receptor in the lesioned basal ganglia is of particular
interest for translating these findings to clinical studies. This could be accomplished by
measuring dopamine D2 receptor binding potential in MPTP mice immediately after the
end of 6 weeks of exercise and at a later time point, such as 4 or 6 weeks after the end
of exercise. When working with the MPTP mouse model of PD in longitudinal studies,
time after the neurotoxin administration is a critical limiting factor of obtaining meaningful
data. It is well established that mice spontaneously recover from MPTP in terms of their
motor performance and striatal markers of dopamine synthesis occurs within 3 months
following an acute lesioning (Jakowec et al 2004b). This is a critical time point that
should be considered when planning longitudinal studies in MPTP mice. Alternatively,
chronic MPTP lesioning protocols may be considered since they provide a longer time
window with greater stability of lesion for longitudinal studies of exercise effects on the
brain.
Another relevant question that needs to be answered in future studies is the
difference between voluntary and forced exercise on dopamine receptor expression in
the basal ganglia. In Chapter 3, only forced treadmill exercise was used, since this type
of exercise provided critical parameters that are important for effects on the brain: the
ability of the researcher to maintain the high intensity and high number of repetitive
movements by keeping the treadmill speed at 20-22 m/min. These parameters are not
controllable when voluntary running wheel is used instead of motorized treadmill.
However, the use of running wheel is not associated with high stress exposure that mice
experience in the motorized treadmill, especially during the first week of running, when
the environment is new and mice are learning to stay away from the foot shock plates by
141
maintaining running speed. The use of aversive motivation stimuli during forced treadmill
training such as foot-shock and/or physical prodding can induce stressful response in
mice and activation of the HPA axis as these two stimuli by themselves are well
documented in the literature to induce elevated levels of CORT in rodents (McEwen
2000). Also, studies have reported increased dopaminergic activity measured by in vivo
voltometry in rats in the striatum and nucleus accumbens upon stressful stimuli, such as
electric shock to the tail or physical restraint (Keller et al 1983; Serrano et al 1989).
While every effort was made to minimize exposure to shock plates in this exercise
protocol by using a metal beaded curtain at the edge of the treadmill belt as a tactile
incentive to prevent them from drifting back onto the shock plates, it is not possible to
exclude aversive motivation stimuli from the protocol for forced treadmill exercise used in
this study that can cause stress response and CORT elevation. Previous studies
showed that acute and chronic stress exposure has effects on dopamine mesolimbic
and nigrostriatal pathways. Studies with rats exposed to 6-hydroxy-dopamine (6-OHDA),
a neurotoxin that selectively kills dopamine producing neurons, showed that after striatal
dopamine loss, a single exposure to 30min tail-shock significantly increases extracellular
dopamine levels in the striatum, as measured by microdialysis (Keefe et al 1990). In the
healthy brain, the nigrostriatal dopaminergic circuit is much less responsive to stressful
stimuli compared to the mesolimbic pathway. However, stress-induced increase in
striatal dopamine levels in 6-OHDA treated rats indicates that in the lesioned state,
nigrostriatal circuits are more sensitive to stress. Another study in rats reported
significant increase of dopamine release in multiple brain regions observed after 30min
tail pinching stress, compared to control animals (Finlay et al 1997). This effect was the
most evident in dopamine projections to the prefrontal cortex, and less in nucleus
accumbens and striatum. Chronic stress has also an effect on the dopaminergic system.
142
Repeated exposure to social stress in adult male rats has an effect on the mesolimbic
dopaminergic system by increasing dopamine transporter (DAT) levels and dopamine
D2 receptor binding potential in the nucleus accumbens in subordinate male rats (Lucas
et al 2004). For these reasons, a future study should delineate between the effects of
stress and motor learning on dopamine D2 receptor expression levels and binding
potential in mice exposed to forced treadmill running.
6.3. Chapter 4: exercise-induced structural plasticity in the basal ganglia
Following the findings from Chapter 3, studies in Chapter 4 further analyzed
exercise-induced neuroplasticity in the injured basal ganglia by focusing on structural
changes on MSNs in the dorsolateral striatum. In this Chapter I report increase in spine
density on MSNs in both control and MPTP treated mice following 6 weeks of high
intensity treadmill exercise, however, the effect was more pronounced in lesioned
animals. At the same time, and in agreement with previous reports, MPTP lesion caused
significant decrease of spine density on MSNs. Additional studies using ultrastructural
electron microscopy showed increased synapse number in the dorsolateral striatum of
MPTP plus exercise mice, and no change in spine area between the treatment groups.
This is the first study to demonstrate exercise-induced changes in spine density and
synapse number outside of the cortical and cerebellar brain areas, using an animal
model of basal ganglia injury. Compared to the motor cortex, cerebellum and
hippocampus, the striatum remains under examined with respect to activity-induced
neuroplasticity, and in particular studies of neuronal morphology. The main reason for
this is the complexity of striatal circuits and functional heterogeneity of MSNs, making
morphological analysis in the striatum difficult to perform and interpret. However, recent
introduction of transgenic mouse lines with fluorescent markers in specific types of
143
neuronal populations in the basal ganglia will likely accelerate experience-dependent
studies of neuroplasticity in the mouse model of PD.
Recent studies using sophisticated fluorescent in vivo imaging techniques
showed that learning a new motor skill is associated with long-term increases in spine
density on cortical pyramidal neurons (Xu et al 2009a; Yang et al 2009). This
observation provides a cellular substrate for re-organization of cortical motor maps
during motor skill training. The question remains whether similar neuronal plasticity
responses are present in animal models of basal ganglia injury, such as the MPTP
mouse. In our study we showed exercise-induced synapse formation in dorsolateral
striatum of MPTP mice. These new synapses could originate from cortical or thalamic
glutamate afferents, and/or from midbrain dopamine projections. A future study using
molecular markers of pre-synaptic terminals could identify the source of new synapse
formation. It is reasonable to hypothesize that activation of motor cortical afferents in
response to high intensity treadmill exercise could drive new synapse formation on
striatal MSNs.
On the other hand, the immediate question that arises from studies in Chapter 4
is whether exercise differentially modulates structural plasticity of MSNs from the direct
and indirect striatal pathways. Also, future studies should investigate a potential
relationship between exercise-induced spine density increase on MSNs and their
electrophysiological properties, such as excitability (measured by changes in amplitude
and frequency of mEPSCs). Studies in Chapter 5 directly focused on this question by
using a transgenic mouse with targeted eGFP expression and biocytin labeling in MSNs
from the indirect pathway.
144
6.4. Chapter 5: exercise modulates neuroplasticity in indirect pathway MSNs
In Chapter 5, our previous studies of exercise-driven alterations in MSNs
morphology were expanded to differentiate between exercise effects on indirect and
direct striatal pathway neurons. Experiments in this chapter focused only on indirect
pathway MSNs and used a transgenic mouse line BAC-Drd2-eGFP. In these mice,
enhanced Green Fluorescent Protein (eGFP) is expressed under the promotor for
dopamine receptor D2. As a consequence, MSNs from the indirect pathway of the basal
ganglia circuits are endogenously labeled with eGFP.
Results presented in this chapter show exercise-induced increase in spine
density on indirect pathway MSNs in the dorsal striatum. In addition,
immunohistochemical analysis showed upregulation of AMPA receptor GluR2 subunits
in these neurons, and this effect was present only in MPTP plus exercise mice
compared to MPTP mice. Also, whole patch recordings from indirect pathway MSNs
showed increased rectification index in response to exercise, again only in MPTP mice.
In contrast to a previous study which examined excitability of MSNs in MPTP mice
following 6 weeks of high intensity exercise (VanLeeuwen et al 2010),
electrophysiological recordings presented in Chapter 5 did not show any significant
differences in sEPSCs, input/output ratio, or in measurements of AMPA/NMDA receptor
ratio in indirect pathway MSNs.
Future studies focusing on analysis of exercise-induced synaptic plasticity of
striatal MSNs morphology and excitability should target on direct pathway MSNs in the
MPTP mouse model (Figure 6.1). This is possible to do using the same transgenic
mouse line Drd2-eGFP, and analyzing non-fluorescent striatal MSNs using the tools of
electrophysiology and biocytin-filled morphology analysis. Alternatively, another
transgenic mouse line is available to address this question, Drd1-eGFP, in which
145
expression of eGFP is restricted to direct pathway MSNs under the control of dopamine
receptor D1. Results presented in this dissertation suggest that both striatal pathways
(including direct and indirect pathway MSNs) are involved in activity-dependent synaptic
plasticity in injured basal ganglia. In addition, to complement studies presented in
Chapter 5 of this dissertation in regards to exercise-induced increase in glutamate
GluR2 receptor subunit expression in striatal MSNs, future studies should focus on early
time point following MPTP-lesion and investigate expression levels of this protein prior to
start of exercise. If our hypothesis is true, and GluR2 receptor subunit is critical for
regulating MPTP lesion-induced corticostriatal hyper-excitability, we can predict that
early following the lesion (for example 5 days post-MPTP) expression levels of GluR2
would be significantly lower in lesioned mice compared to the controls, and that
exposure to high-intensity treadmill exercise promotes normal levels of GluR2
expression in striatal MSNs. Together, these studies will provide a comprehensive
analysis of exercise-induced neuroplasticity in the striatum in regards to MSNs
morphology, excitability and expression of glutamate AMPA receptors that provide a
critical mechanism of activity-dependent synaptic plasticity in injured basal ganglia.
146
Figure 6.1: Future directions in studying effects of exercise-induced synaptic plasticity in
striatal pathways of injured basal ganglia. Analysis of morphology and excitability of
direct pathway MSNs (upper panels) in the MPTP mouse model will complement studies
presented in this dissertation. In addition, a time course study of expression levels of
GluR2 receptor subunit (for example early after MPTP lesion and before starting
treadmill exercise) will help with our better understanding of the role of glutamate
receptors in activity-dependent synaptic plasticity in injured basal ganglia.
Still, many questions remain open regarding the molecular and cellular
mechanisms of basal ganglia plasticity in response to treadmill exercise. A critical
mechanism that should be investigated in future studies in the role of brain-derived
neurotrophic factor (BDNF) on morphology and glutamate receptor expression in striatal
MSNs in response to exercise. Previous studies showed that exercise upregulates
BDNF expression in multiple brain regions, including the hippocampus and striatum
(Adlard et al 2005; Bakos et al 2009; Gomez-Pinilla et al 2002; Marais et al 2009; Oliff et
Direct pathway (dopamine D1 receptor)
Indirect pathway (dopamine D2 receptor)
147
al 1998; Strasser et al 2006; Vaynman et al 2004; Vaynman et al 2006; Widenfalk et al
1999). The role of exercise-driven activity of BDNF in injured basal ganglia has not been
studied in detail and could provide a cellular and molecular mechanism for the benefits
of exercise reported in our studies. These future studies should take advantage of two
transgenic mouse lines that have differential expression of BDNF in the brain. The first
line is a BDNF +/- hemizygote knock-down that has only one copy of the bdnf gene
functional, resulting in mice that have 50% less BDNF protein in the brain. The second
line is a BDNF overexpressor, which has human bdnf gene expressed under regulation
of actin promotor, resulting in mice that have constitutively high levels of BDNF protein in
the brain. Studies of exercise-induced neuroplasticity in the striatal MSNs in these
transgenic mice can start answering important questions regarding the molecular
mechanisms that link physical activity, such as treadmill exercise, and morphological
and physiological changes we observed in striatal MSNs.
6.5. Chapters 2-5: the big picture
Studies presented in this dissertation contribute to our increasing
knowledge and understanding of brain plasticity mechanisms in health and
neurodegenerative diseases. Understanding these mechanisms in detail is important as
they can be used to improve current therapies for neurodegenerative diseases, and to
design prevention strategies against these diseases as well. Using the MPTP mouse
model of basal ganglia injury, results of the studies presented in this dissertation can be
used to develop a model of molecular and cellular mechanisms involved in exercise-
induced synaptic plasticity in injured basal ganglia (Figure 6.2).
The ultimate long-term goal of this doctoral thesis project was to contribute to
better understanding of the MPTP mouse model of PD, and to identify molecular targets
148
within the injured basal ganglia that trigger neuronal plasticity producing beneficial
behavioral and neuronal outcomes. The results presented in this doctoral thesis highlight
the value of preclinical research in animal models of dopamine depletion and the
importance of translational research for providing both rationale and a means for future
studies in individuals with PD.
Figure 6.2: The big picture: overview of molecular and cellular mechanisms underlying
exercise-induced neuroplasticity in the basal ganglia based on experimental results
presented in this dissertation. Compared to healthy basal ganglia (A), severe dopamine
loss in the striatum of MPTP mice (B) impairs indirect pathway MSNs causing dendritic
spine loss, and decreased expression of dopamine D2 receptor, as well as increased
glutamate corticostriatal neurotransmission (represented by enlarged gray axon making
a synapse onto the green MSN in the middle panel). Spine loss on MSNs could
potentially be accompanied by loss of corticostriatal synapses. (C) Exercise-induced
upregulation of GluR2 glutamate receptor subunit is present in MSNs from both
pathways in the cell body (measured by increased immunoreactivity using a specific
antibody) and on synapses (indicated by an increase in rectification index). Direct
pathway MSN is presented in orange and indirect pathway MSN is green; purple star
represent dopamine D2 receptor expression on indirect pathway MSN and axon
terminals of motor cortex neurons (in grey); red color represents GluR2 receptor subunit
expression on MSNs; blue represents cell nucleus.
It is very well accepted that physical exercise as part of treatment of patients with
PD has beneficial effects on ameliorating some of the motor symptoms. However, at the
A B C
149
moment there is no agreement among researchers and health specialists about the kind
of exercise that is the most beneficial, what is the optimal length of exercise intervention,
and how long do the benefits last. More studies focusing on the beneficial effects of
exercise are needed both in humans and animal models of PD. Preliminary animal and
human studies indicate that intervention early in disease is far more effective than late.
For human studies this means that newly diagnosed patients would experience
the most benefit from exercise program that starts as soon as the first symptoms are
present. Some animal studies suggest the possibility that the integrity of the injured
basal ganglia strongly depends on maintaining a minimum level of motor activity through
life. So, in addition to the early start of exercise intervention, long-term maintenance of
basal physical activity is an important factor to take into consideration.
When designing animal studies to investigate the beneficial role of exercise in the
recovery from basal ganglia injury, an important consideration to keep in mind is that
exercise by itself is neuroprotective for the basal ganglia. Taking into consideration that
the established rodent models of PD are produced by acute or chronic administration of
neurotoxins, exposing these animals to exercise prior to neurotoxin treatment could
severely impact results in different outcome measures. Optimal time to start with
exercise in rodent models of PD would be early following the onset of injury, however
after the cell death caused by neurotoxins is complete.
150
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APPENDIX:
ADDITIONAL PUBLICATION LIST
Gorton LM, Vuckovic MG, Vertelkina N, Petzinger GM, Jakowec MW, Wood RI. (2010)
Exercise effects on motor and affective behavior and catecholamine neurochemistry in
the MPTP-lesioned mouse. Behav Brain Res. 213(2):253-62.
Petzinger GM, Vanleeuwen JE, Walsh JP, Akopian GK, Vuckovic M, Jakowec MW
(2010) Enhancing Neuroplasticity in the Basal Ganglia: The Role of Exercise in
Parkinson's Disease. Mov Disord. H 24(777):2585
Vanleeuwen JE, Petzinger GM, Walsh JP, Akopian GK, Vuckovic M, Jakowec MW.
(2010) Altered AMPA receptor expression with treadmill exercise in the 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse model of basal ganglia injury. J
Neurosci Res. 88(3):650-668
Petzinger GM, Walsh JP, Akopian G, Hogg E, Abernathy A, Arevalo P, Turnquist P,
Vucković M, Fisher BE, Togasaki DM, Jakowec MW. (2007) Effects of treadmill
exercise on dopaminergic transmission in the 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine-lesioned mouse model of basal ganglia injury. J Neurosci.
27(20):5291-300
Abstract (if available)
Abstract
The mammalian brain is a remarkable organ that continues to fascinate biologists with its ability to undergo experience-dependent adaptations, and this property is preserved throughout the life. Brain plasticity on the molecular and cellular level within neurons is thought to occur in response to neuronal activity, continuously changing the strength of synapses and thus influencing the connections between neurons. Synapses are weakened or strengthened in response to specific patterns of neuronal activity. The underlying mechanism of synaptic plasticity is thought to consist of the regulated release of neurotransmitters (chemical mediators of neuronal communication) from the presynaptic side, and changes in the expression levels and availability of corresponding receptors located predominantly on the postsynaptic side of the synapse. Numerous studies over the past three decades have focused on elucidating the molecular basis of synaptic plasticity, with the goal of better understanding the link between transient changes in neuronal activity in response to experience, and short- and long-term changes in brain circuitry that underlie learning, memory and adaptive behavior. However, many key questions remain unresolved. The results of research efforts presented in this dissertation represent a modest contribution to our increasing knowledge of experience-dependent brain plasticity and its relevance to human health.
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Vučković, Marta
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Neuroplasticity of the basal ganglia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease
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