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INVESTIGATION OF THE EFFICACY OF
VARIOUS NEUROPROTECTION AGENTS FOR
THEIR POTENTIAL USE IN THE TREATMENT
OF PARKINSON’S DISEASE

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Kelly Lynn Janis

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INVESTIGATION OF THE EFFICACY OF VARIOUS
NEUROPROTECTION AGENTS FOR THEIR POTENTIAL USE IN THE
TREATMENT OF PARKINSON’S DISEASE

BY

Kelly Lynn Janis

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Neuroscience

2008

ABSTRACT

INVESTIGATION OF THE EFFICACY OF VARIOUS NEUROPROTECTION
AGENTS FOR THEIR POTENTIAL USE IN THE TREATMENT OF PARKINSON’S
DISEASE

BY
Kelly Lynn Janis

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the
progressive loss of the nigrostriatal dopamine (N SDA) neurons. The loss of these
neurons leads to several debilitating symptoms including slowness of movement, rigidity,
postural instability, and resting tremor. Current therapies for PD are aimed at replacing
DA deficits for symptomatic relief, but treatments to slow or halt the progressive loss of
NSDA neurons are not yet available. Therefore, the primary goal of this dissertation was
to examine potential neuroprotective therapies using rodent models of PD.

Several potential etiologies of PD have been proposed including environmental
exposure to neurotoxins, oxidative stress, inflammation, mitochondrial dysfunction, and
protein aggregation. The neuroprotective strategies explored in this dissertation were
targeted to reduce one or more of these causes of PD, induce the development of new
NSDA neurons, or explore the effects of a lack of specific functional proteins in either a
neurotoxin or inflammatory model of PD. The potential neuroprotective effect of the
drug sildenafil in the neurotoxin MPTP (1 -methy1-2-phenyl-1 ,2,3,6-tetrahydropyridine)
model of PD was examined. However, sildenafil failed to demonstrate neurogenesis or a
neuroprotective effect as indicated by a lack of attenuation of the loss of NSDA cell

bodies and axon terminals following MPTP treatment.

Increased inflammation and oxidative stress observed in models of PD are
primarily mediated by microglial cells within the brain. These two processes contribute
to the progression of PD and partially result from activation of the microglial enzyme
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Inhibition of NADPH
oxidase using the inhibitor apocynin was tested as a potential neuroprotective drug in the
MPTP model of PD but was found to be ineffective in attenuating MPTP induced
activation of NADPH oxidase and loss of NSDA axon terminals.

Endogenous cannabinoids (ECB) are involved in the regulation of NSDA neurons
and microglial inflammation. NSDA neuronal integrity, activity, and regulation were
initially characterized in mice lacking functional cannabinoid (CB) 1 and CB2 receptors,
to determine if these mice were comparable to wildtype (WT) control mice. Mice
lacking CB1 and CB2 receptors were determined to have typical integrity, activity and
regulation of NSDA neurons. Next, CB1/CB2 knockout (KO) mice were used to
determine if absence of CB1 and CB2 receptors alters NSDA neuronal susceptibility to
Nfl’TP neurotoxicity or lipopolysaccharide (LPS) induced acute inflammation. The loss
of axon terminal DA concentrations was found to be attenuated in a MPTP model of PD
suggesting inhibition of one or more of these receptors could be neuroprotective in PD.
However, pharmacologic inhibition of these receptors (either individually or together)
failed to replicate the neuroprotective results seen in CBl/CB2 KO mice. In addition,
acute inflammation induced by LPS did not alter the integrity or activity of NSDA
neurons. These findings suggest that neither sildenafil or apocynin are viable
neuroprotective agents in the treatment of PD, and that CB1 and CB2 receptors may play

a role in MPTP induced loss of NSDA neurons.

This dissertation is dedicated to my Mom and Dad,
thank you for all of your support and encouragement through out the years,
and Mom for all of the times you listened to me,
and to Dr. Susan Aronica, my long time mentor for encouraging me to pursue this degree

afler I discovered my love of scientific research when I worked in your lab

iv

ACKNOWLEDGEMENTS

I would like to thank first my Mom and Dad who helped me get to this point in
my life, Steve and Sarah for your love and encouragement, Matthew, Noah, and Anne
who always remind me of the simple joys in life, and my many other family members
who are to many to name for their support, and Lauren and Margaret for always being
there for me you will always be my best friends.

I would like to thank my advisors Drs. Keith Lookingland and John Goudreau for
their guidance and dedication to my scientific training over the past few years. I will
always be thankful to have had the opportunity to be trained by you and be a member of
your lab.

I would also like to thank my committee members Drs. Patricia Ganey, Barbara
Kaplan, and David Kreulen for their help and advisement on my dissertation.

I would like to thank the Neuroscience and College of Osteopathic Medicine Dual
Degree Pro grams’ faculty for your support and mentorship you provided in my training,
and also all my fellow students and especially my friends who were always there for me
during the many steps it took to reach this point.

Lastly I would like to thank my fellow lab members who made my experience in
the lab one I will never forget. The collaborative and fun environment you created both
past and present, and thoughtful scientific input during my training made this journey one

I will always be thankful for.

TABLE OF CONTENTS

List of Tables ............................................................................. x
List of Figures ............................................................................ viii-xv
List of Abbreviations ................................................................... xvi-xix
Chapter 1. Introduction ............................................................... 1
A. Statement of Purpose ....................................................... 1
B. Causes of PD ................................................................. 3
C. The Basal Ganglia ........................................................... 6
D. DA Synthesis, Release, and Metabolism ................................. 9
E. DA Neuronal Pepulations .................................................. 15
F. DA Receptors ................................................................ 18
G. Regulation of DA Synthesis in Central Dopaminergic Neurons ...... 21
H. Indices of DA Neuronal Integrity and Activity .......................... 24
I. Serotonin Synthesis and Neuronal Function .............................. 26
J. Neurotoxic Models of PD ................................................... 28
K. Inflammation in the Brain ................................................... 34
L. Neurogenesis and Sildenafil ................................................ 38
M. The Role of Nicotinamide Adenine Dinucleotide Phosphate
(NADPH) Oxidase in Microglial Mediated Death of NSDA. . . . . ......39
Neurons
N. Cannabinoids and Endocannabinoids ..................................... 42
O. Dissertation Objectives ...................................................... 51
Chapter 2. Materials and Methods ................................................... 53
A. Animals ........................................................................ 53
B. Drugs ........................................................................... 61
C. Brain Tissue Preparation .................................................... 68
D. Assay of Amines, Amine Metabolites, and Apocynin in
Brain Tissue .................................................................. 70
E. cGMP Assay .................................................................. 79
F. Plasma Sildenafil Determination .......................................... 81
G. Western Blot Analysis ...................................................... 82
H. Immunohistochemistry and Stereology Cell Counts .................... 90
I. NADPH Oxidase Activity Assay .......................................... 92
J. Mitochondrial Complex I Assay ........................................... 94
K. MPP+ Assay .................................................................. 98
L. Statistical Analysis ........................................................... 102

vi

Chapter 3. Effects of Sildenafil on N igrostriatal Dopamine Neurons in a

Murine Model of Parkinson’s Disease ...................................... 103
A. Introduction ................................................................... 103
B. Materials and Methods ...................................................... 107
C. Results ......................................................................... 109
D. Discussion ..................................................................... 118

Chapter 4. Apocynin as a Neuroprotective Agent in the MPT P Model

of PD ............................................................................... 123
A. Introduction ................................................................... 123
B. Materials and Methods ...................................................... 128
C. Results ......................................................................... 130
D. Discussion ..................................................................... 153

Chapter 5. The Characterization of the Neurochemical Properties and
Neuromodulation of Dopaminergic Neurons in Wildtype and

Cannabinoid 1 and 2 Receptor Knockout Mice ........................... 160
A. Introduction ................................................................... 160
B. Materials and Methods ...................................................... 166
C. Results ......................................................................... 168
D. Discussion ..................................................................... 191
Chapter 6. The Effects of a Lack of Cannabinoid Receptors in the MPTP
Model of PD ...................................................................... 203
A. Introduction ................................................................... 203
B. Materials and Methods ...................................................... 208
C. Results ......................................................................... 210
D. Discussion ..................................................................... 252
Chapter 7. Lipopolysaccharide Activation of Microglia as an Inflammatory
Model of PD in Wildtype and CBl/CB2 Receptor KO Mice ........... 262
A. Introduction ................................................................... 262
B. Materials and Methods ...................................................... 268
C. Results ......................................................................... 270
D. Discussion ..................................................................... 281
Chapter 8. Concluding Remarks ...................................................... 286
References ................................................................................. 294

vii

Table #

2-2

2-3

3-1

4-1

4-2

4—3

6-1

6—2

6-3

LIST OF TABLES

Title

PCR primers for CB1 and CB2 receptors and CB2 KO

neomycin cassette ..................................................

MPTP treatment paradigms ......................................

Primary and secondary antibodies used for Western

blotting ...............................................................

Numbers of TH immunoreactive cells in the unilateral
substantia nigra pars compacta of sildenafil and/or MPTP

treated mice .........................................................

Neurochemical concentrations in the nucleus accumbens

following sub-chronic MPTP and apocynin treatment. . . . . . . .

Neurochemical concentrations in the median eminence

following sub-chronic MPTP and apocynin treatment ........

Neurochemical concentrations in the nucleus accumbens

following repeated acute MPTP and apocynin treatment. . ..

Parkin and DARPP-32 protein in the striatum following

acute MPTP treatment .............................................

Effects of sub-chronic MPTP on DA, DOPAC and the
DOPAC ratio in the nucleus accumbens and median

eminence of rimonabant or SR144528 treated mice ..........

Effects of sub-chronic MPTP on DA, DOPAC and the
DOPAC ratio in the nucleus accumbens and median

eminence of rimonabant and SR144528 treated mice ........

viii

Page #

....56

..... 62

....89

..... 117

143

....144

...... 146

..... 218

..... 246

...... 247

Figure #
1-1
1-2

1-3

1-4
1-5
1-6

1-7

1-8

1-9

1-12

2-1

2-2

2-3

LIST OF FIGURES

11ng bgei
Anatomy of the basal ganglia ........................................... 7
TH synthesis of DOPA from dietary tyrosine ....................... 12
DA synthesis, release, reuptake, and metabolism at the

NSDA axon terminal ................................................... 13
DA is metabolized by one of two pathways to HVA ............... 14

Catecholaminergic neuronal populations in the rodent brain. . 1 7

Feedback regulation of NSDA neuronal activity .................... 23
Metabolism of MPTP to MPP+ and transport of MPP+

into DA neurons ......................................................... 30
Effects of MPP+ at the dopaminergic neuronal terminal ........... 31

Direct and indirect activation of microglia induces
microgliosis in models of PD .......................................... 36

NADPH oxidase activation and production of superoxide
in microglial cells ........................................................ 40

Depolarization induced suppression of neurotransmission
by cannabinoids .......................................................... 46

CB1 receptor expression within the basal ganglia ................... 48

CB1 receptor product in WT mice, but not CB1/C82 KO
mice ........................................................................ 58

CB2 receptor product in WT mice, but not CB1/CB2 KO
mice ........................................................................ 59

Apocynin voltamogram .................................................. 71

Diagram showing striatum and nucleus accumbens sampling
sites ......................................................................... 73

ix

2—5

2-7

2-8

2-9

2-10

2-11

2-12

3-1

3-2

3-3

3—5

3-6

4-1

Chromatograms show peak height and retention time for the
respective neurochemicals or apocynin ................................ 77

Chromatograms showing peak height and retention times
for DA, DOPAC and apocynin in striatum samples .................. 78

cGMP standard curve ..................................................... 80

Pictorial diagram of striatum and ventral midbrain tissue sites
taken by tissue punch or dissection ..................................... 83

Pictorial diagram of sucrose gradient fractionation of membrane
and cytosolic fractions .................................................... 85

Pictorial diagram of isolation of membrane and cytosolic
fractions using ultracentrifugation ....................................... 86

Plot of striatum brain tissue protein content verses the difference
in absorbance decay before and afier Complex I inhibition ......... 97

Standard curve of MPP+ concentrations'measured using liquid
chromatography mass spectrometry .................................... 101

Plasma free sildenafil and brain cGMP levels after sildenafil
administration .............................................................. 1 1 1

Effects of chronic MPTP administration on DA and DOPAC
concentrations, and the DOPAC/DA in the striatum of mice
pro-treated with sildenafil ................................................ 112

Effects of chronic MPTP administration on DA and DOPAC
concentrations, and the DOPAC/DA in the striatum of
concurrent sildenafil treated mice ....................................... 113

Effects of chronic MPTP administration on DA and DOPAC
concentrations, and the DOPAC/DA in the striatum of

post-treatment sildenafil treated mice .................................. 114
Effect of chronic MPTP administration on TH protein content

in the striatum of mice pre-treated with sildenafil .................... 115
Effects of chronic MPTP administration on TH immunoreactive

cells in the substantia nigra ............................................. 116
Measurement of apocynin in the brain using HPLC-EC ............. 133

4—2

4-3

4-4

4-8

4-9

4-10

4-11

5-2

5-3

5-5

5-6

Apocynin decreases the concentrations of DOPAC and the

DOPAC/DA ratio in the striatum ........................................ 134
Apocynin dose dependently decreases DOPAC concentrations

and reduces the DOPAC/DA ratio in the striatum ..................... 135
Apocynin does not inhibit MAO B in vitro ............................ 136

Apocynin does not alter MPTP induced DA depletion in the
striatum ..................................................................... 140

Apocynin does not alter MPTP induced HVA depletion in
the striatum ................................................................. 141

Membrane bound p67Phox protein in the striatum ................... 142

NADPH oxidase activation following repeated acute MPTP
treatment .................................................................... 146

Apocynin does not alter repeated acute MPTP induced DA
depletion in the striatum .................................................. 149

Loss of TH protein content in the striatum with repeated
acute MPTP treatment is not attenuated with apocynin .............. 150

Apocynin does not reduce p67Phox translocation to the
membrane or an increase in gp91Phox subunit membrane
expression in the striatum observed with repeated acute MPTP

treatment .................................................................... 15 1
C81 receptor expression within the basal ganglia .................... 161
WT and CBl/CB2 KO mouse body weight ........................... 167

Concentrations of DA and DOPAC and the DOPAC/DA ratio
in the striatum of male WT and CB1/C82 KO mice ................. 170

Concentrations of HVA and the HVA/DA ratio in the striatum
of WT and CB1/CB2 receptor KO mice ............................... 171

Expression of TH, phosphorylated serine 40 TH, and DAT
in the striatum of WT and CB1/CB2 KO mice as determined
by Western blot ............................................................ 172

TH immunoreactive neurons within the substantia nigra of
WT and CBl/CB2 KO mice ............................................. 173

xi

5-7

5-8

5-13

5-14

5-15

5-16

5—17

5-18

6-1

6-2

Bilateral TH cell body stereological counts in the substantia
nigra and TH protein expression in the ventral midbrain of
WT and CBl/CB2 KO mice ............................................. 174

Concentrations of DA and DOPAC and the DOPAC/DA
ratio in the nucleus accumbens of WT and CBl/CB2 KO
mice ......................................................................... 176

Concentrations of HVA and the HVA/DA ratio in the
nucleus accumbens of WT and CB1/CB2 KO mice .................. 177

Concentrations of DA and DOPAC and the DOPAC/DA ratio
in median eminence of WT and CB1/CB2 KO mice ................. 178

D2 autoreceptor regulation in the striatum of WT and
CB1/CB2 KO mice ....................................................... 181

D2 autoreceptor regulation in the nucleus accumbens of
WT and CB1/CB2 KO mice ............................................. 182

D2 autoreceptor regulation in the median eminence of
WT and CBl/CB2 KO mice ............................................. 183

DA receptor regulation in the striatum of WT and
CB1/CB2 KO mice ....................................................... 185

DA receptor regulation in the nucleus accumbens of WT
and CB1/CB2 KO mice .................................................. 186

DA receptor regulation in the median eminence of WT
and CB1/CB2 KO mice .................................................. 187

Concentrations of 5HT, SHIAA, and the SHIAA/SHT ratio
in the striatum of WT and CB1/CB2 KO mice ....................... 189

Concentrations of 5HT, SHIAA, and the SHIAA/SHT ratio
in the nucleus accumbens of WT and CBl/CB2 KO mice ......... 190

Schematic depicting the location of C31 receptors within
the basal ganglia .......................................................... 204

Time course effects of acute MPTP treatment on DA,

DOPAC and the DOPAC/DA ratio in the striatum of
WT and CB1/CB2 KO mice ............................................. 212

xii

6-3

6-4

6-5

6-8

6-9

6-10

6-11

6-12

6-13

6-14

Time course effects of acute MPTP treatment on HV A and

the HVA/DA ratio in the striatum of WT and CBl/CB2 KO
mice ........................................................................ 213

Lack of an effect of acute MPTP treatment on TH and DAT
protein content in the striatum of WT and CB1/CB2 KO ........... 216

Alpha-synuclein and p67Phox cytosolic protein content in
the striatum of WT and CB1/CB2 KO mice over time
following saline or MPTP treatment .................................... 217

DARPP-32 and Parkin protein expression in the striatum in
WT and CBl/CB2 KO mice following saline or MPTP
Treatment .................................................................. 219

Time course effects of acute MPTP treatment on DA,
DOPAC and the ratio of DOPAC/DA in the nucleus
accumbens of WT and CB1/C82 KO mice ........................... 221

Time course effects of acute MPTP treatment on HV A
and the ratio of HVA/DA in the nucleus accumbens of WT
and CB1/C32 KO mice .................................................. 222

Time course effects of acute MPTP treatment on DA,
DOPAC and the ratio of DOPAC/DA in the median
eminence of WT and CB1/CB2 KO mice ............................. 224

Time course effects of acute MPTP treatment in the striatum
of WT and CB1/CB2 KO mice .......................................... 226

Time course effects of acute MPTP treatment in the nucleus
accumbens of WT and CB1/C32 KO mice ........................... 227

Effects of sub-chronic MPTP treatment on DA, DOPAC and
the ratio of DOPAC/DA in the striatum of WT and
CB1/C32 KO mice ....................................................... 231

The effect of sub-chronic MPTP treatment on TH and
phosphorylated serine 40 TH protein in the striatum of WT
and CB1/CB2 KO mice .................................................. 232

TH immunoreactive cells within the substantia nigra of WT
and CB1/CB2 KO mice following sub-chronic MPTP

treatment ................................................................... 233

xiii

6-15 Bilateral TH cell counts in the substantia nigra and ventral
midbrain TH protein content in WT and CB1/CB2 treated
with MPTP using the sub-chronic treatment paradigm .............. 234

6-16 Effects of sub-chronic MPTP treatment on DA, DOPAC
and the ratio of DOPAC/DA in the nucleus accumbens of
WT and CB1/CB2 KO mice ............................................. 237

6-17 Effects of sub-chronic MPTP treatment on DA, DOPAC
and the ratio of DOPAC/DA in the median eminence of
WT and CB1/CB2 KO mice ............................................. 238

6-18 Lack of an effect of blockade of either CB1 or CB2
receptors on MPTP induced changes in DA, DOPAC and
the ratio of DOPAC/DA in the striatum of WT mice ............... 242

6-19 Lack of an effect of blockade of both CB1 and CB2
receptors on MPTP induced changes in DA, DOPAC and
the ratio of DOPAC/DA in the striatum of WT mice ............... 243

6-20 Mitochondrial complex I activity in the striatum of
MPTP treated WT and CB1/C32 KO mice ........................... 250

6-21 Time course effects of a single injection of MPTP on the
concentrations of MPP+ and DA and the DOPAC/DA ratio
in the striatum of WT and CBl/CB2 KO mice ...................... 251

7-1 Ipsilateral and contralateral concentrations of DA and
DOPAC and the DOPAC/DA ratio in the striatum of mice
injected unilaterally with PBS or LPS into the substantia
nigra ....................................................................... 272

7-2 TH cell numbers in the contralateral and ipsilateral
substantia nigra of mice injected unilaterally with either
PBS or LPS into the substantia nigra ................................. 273

7-3 TH protein content in the contralateral and ipsilateral
substantia nigra of mice injected with either PBS or LPS ......... 274

7-4 Cytosolic p67Phox protein content in the contralateral
and ipsilateral substantia nigra of mice injected with either
PBS or LPS ............................................................... 275

7-5 Concentrations of DA, DOPAC, and the DOPAC/DA ratio

in the striatum of WT and CBl/CB2 KO mice following
peripheral LPS administration ........................... fl .............. 278

xiv

7-7

Concentrations of DA, DOPAC, and the DOPAC/DA ratio
in the nucleus accumbens of WT and CBl/CB2 KO mice
following peripheral LPS administration ............................ 279

Concentrations of DA, DOPAC, and the DOPAC/DA ratio

in the median eminence of WT and CB1/CB2 KO mice
following peripheral LPS administration ............................ 280

XV

Z—AG

3 —MT

SHIAA

SET

6 - OHDA

LIST OF ABBREVIATIONS

2-arachidonoylglycerol
3-methoxytyramine
5-hydroxyindoleacetic acid

serotonin (5-hydroxytryptamine)
6-hydroxydopamine

aldehyde dehydrogenase

anandamide membrane transporter
adenine triphoshphate

brain derived neurotrophic factor
dihydrobiopterin

tetrahydrobiopterin

3 ’,5 ’-cyclic adenosine monophosphate
cannabinoid

cannabinoid 1 receptor

cannabinoid 2 receptor
catechol-O-methyltransferase
3’,5’-cyclic guanosine monophosphate
dopamine

DA and CAMP regulated phosphoprotein
dopamine transporter

DOPA decarboxylase

xvi

DOPA
DOPAC
DQPAL
ECB
ED
FAAH
GABA
GAPDH
GEL
GPe
GPi

HP LC-EC

HVA
HVAA
IL— 1 [3
IL-6
iNos
KO
L‘DOPA
LC-Ms
LPS
MAO B
IV11313P+

3,4-dihydroxyphenylalanine
3,4-dihydroxyphenylacetic acid
dihydroxyphenylacetaldehyde
endocannabinoid

erectile dysfimction

fatty acid amide hydrolase
gamma-aminobutyric acid
glyceraldehyde-3-phosphate dehydrogenase
gamma-butyrolactone

globus pallidus external segment
globus pallidus internal segment

high performance liquid chromatography with electrochemical
detection

homovanillic acid

homovanillic acid aldehyde

interleukin-1 beta

interleukin-6

inducible nitric oxide synthase

knockout

3,4-dihydroxy-L-phenylalanine

liquid chromatography coupled to mass spectrometry
lipopolysaccharide

monoamine oxidase B

1 -methyl-4-phenyl-2,3 -dihydropyridine

xvii

MLDA
IVIPP+
MPTP
NADPH
N FKB

N SDA

mesolimbic dopamine

1 -methyl-4-phenylpyridinium

1 -methyl-phenyl- 1 ,2,3 ,6-tetrahydropyridine
nicotinamide adenine dinucleotide phosphate
nuclear factor kappa beta
nigrostriatal dopamine

phosphate buffered saline

oxygen

polymerase chain reaction
Parkinson’s Disease
phosphodiesterase 5

reactive oxygen species

substantia nigra pars compacta
substantia nigra pars reticulate
synaptosomal-associated protein 25
subthalamic nucleus

subventricular zone

tyrosine hydroxylase
tuberoinfindibular

toll like receptor-4

tumor necrosis factor alpha
Wildtype

ventral tegrnental area

xviii

VMATZ vesicular monoamine transporter-2

xix

Chapter 1: General Introduction

A- Statement of Purpose

Parkinson’s disease (PD) is a neurodegenerative disorder resulting from the loss
0 f the nigrostriatal dopamine (N SDA) neurons. NSDA neuronal cell bodies are present
i n the substantia nigra pars compacta (SNpc) of the ventral midbrain and their axonal
processes terminate in the striatum. NSDA neurons are important in motor control, and
the loss of these neurons is associated with the development of the classic symptoms of
PD, which include resting tremor, rigidity, slowness of movement, and postural
instability. The compensatory increase in activity that occurs in the remaining NSDA
neurons likely prevents the symptoms of PD from being detected until 50-60% of the
neurons have been lost.
Currently there is no neuroprotective treatment available to slow or halt the
pro gression of PD upon diagnosis, rather only symptomatic treatments exist.
S mptomatic treatments consist of administration of the DA precursor 3,4-dihydroxy-L-
Phenylalanine (L-DOPA) which is converted to DA, DA agonists, or monoamine oxidase
B CIVIAO B) inhibitors which block the metabolism of DA to the metabolite 3,4-
dil“lydroxyphenylacetic acid (DOPAC). Therefore, the elusive goal of a neuroprotective
treatment for PD is still sought after.
The purpose of this dissertation is to test certain potential neuroprotective
Strategies for PD using neurotoxic and inflammatory models of the disease. The
therapeutic potential of pharmacological agents or genetic deletion of specific genes was

evaluated by measuring DA neuronal cell body and axon integrity, activity of

dopaminergic neurons, and microglial activation following neurotoxic or inflammatory
insult. If a potential drug or genetic knockout of a gene is considered neuroprotective,

N SDA neuronal integrity will be either partially or completely maintained.

3- Causes of PD

Several possible causes of idiopathic PD have been proposed including exposure
to environmental toxins, mitochondrial dysfimction, oxidative stress, protein misfolding
and aggregation, and inflammation related damage to NSDA neurons (Dauer and

Przedborski, 2003). These potential causes of PD usually are interconnected with one or

more of the others.

The environmental toxin hypothesis is associated with studies that have shown
humans in rural areas exposed to pesticides have a higher incidence of PD (Gorell et al.,
1 998; Priyadarshi et al., 2001). Direct dopaminergic neurotoxins are also implicated in

the progressive loss of NSDA neurons following a brief exposure to the relatively
se lective neurotoxin 1-methyl—4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Langston et
al - , 1983). Although MPTP may initiate death of NSDA neurons acutely, the progressive
loss of NSDA neurons long after MPTP is cleared fi'om the system is attributed to
inflammation and subsequent oxidative stress within the substantia nigra (Langston et al.,
1 999).

Mitochondrial dysfunction can be incredibly detrimental to NSDA neurons and
leads to oxidative stress. The discovery of the mitochondrial Complex I inhibitors MPTP
and rotenone (which induce the death of NSDA neurons) fielded the investigation of this
cause of PD (Langston et al., 1983; Betarbet et al., 2000). Inhibition of the mitochondrial
respiratory chain prevents the formation of adenosine triphosphate (ATP) by blocking the
t1‘El-nsfer of electrons down the chain to molecular oxygen. Reduced ATP production
leads to energy depletion within the cell and reactive oxygen species (ROS) formation

(Dauer and Przedborski, 2003). ROS species such as superoxide, hydroxyl radical, and

hydrogen peroxide can react with the reactive nitrogen species nitric oxide and produce
the product peroxynitrite which inhibits the mitochondrial respiratory chain (Tieu et al.,
2 003; Tretter et al., 2004). ROS react with cellular proteins, DNA and lipids inducing
cellular dysfunction. ROS also react with cytosolic DA forming DA-quinones which
re act with cellular proteins inducing further cellular damage (Asanuma et al., 2004). A
reduced ability of NSDA neurons to combat oxidative stress can also contribute to the
s elective loss of NSDA neurons compared to other dopaminergic neuronal populations
(Drechsel and Patel, 2008).
Protein misfolding and inclusion formation such as cytoplasmic Lewy bodies in
N SDA neurons which contribute to the progression of PD could result from one of many
causes. Age leads to a reduced ability to catabolize misfolded proteins which may
contribute to an increase in inclusion formation seen with increasing age (Sherman and
Goldberg, 2001). Damage of cellular proteins by ROS can contribute to an increase in
dalmaged or misfolded proteins (Giasson et al., 2000). Altered proteosome function (the
Ce 1 lular unit which degrades proteins) or the pathway that leads to final degradation of
PI‘Oteins also contributes to build up of cytoplasmic inclusions (Tanaka et al., 2001;
1)etrucelli et al., 2002). Mutations in genes comprising part of the protein degradation
Pathway have been associated with genetic causes of PD, indicating the importance of
appropriate handling of misfolded proteins (Dauer and Przedborski, 2003).
Inflammation is also implicated in the death of NSDA neurons and may
contribute to the progressive nature of the disease. Activation of microglial cells, the
r esident immune cells in the brain, either directly through the death of NSDA neurons or

ll"(lirectly through inflammatory activation of microglia can lead to the production of

 

ROS. and reactive nitrogen species, and release of pro-inflammatory cytokines (Block and
H ong, 2005). These factors lead to direct inflammatory mediated death of NSDA
neurons and/or contribute to the recruitment of more micro glial cells that perpetuate the
inflammatory process. The greater number of microglia found in the ventral midbrain
containing the substantia nigra, and the reduced ability of NSDA neurons to deal with
oxidative stress likely contributes to a more selective inflammatory mediated loss of these

neurons (Kim et al., 2000; Block and Hong, 2005; Drechsel and Patel, 2008).

C. The Basal Ganglia

The NSDA neurons are part of the basal ganglia network which controls motor
output by the thalamus. The basal ganglia consists of two separate pathways; direct and
indirect (Figure 1-1). Cortical neurons originating in the motor segment of the cerebral
cortex project to the striatum and release the excitatory neurotransmitter glutamate onto
medium spiny neurons (Squire, 2003). Medium spiny neurons are components of the
stri atonigral and striatalpallidal segments and the direct and indirect pathways,
respectively.

As shown in Figure 1-1, the direct pathway begins with the projection of the
stri atonigral neurons from the striatum to the substantia nigra pars reticulata (SNpr) and
inteI‘nal segment of the globus pallidus (GPi) and release the inhibitory neurotransmitter
gamma-aminobutyric acid (GABA) and the opioid peptides dynorphin and substance P
(Steiner and Gerfen, 1998). GABAergic neurons project from the SNpr and GPi to the
thalatnus. The thalamus projects glutamatergic neurons onto the motor cortex facilitating
movement (Squire, 2003). The direct pathway leads to the facilitation of movement since
its activation causes inhibition of GABAergic neurons firing onto the thalamus
(Ben arroch, 2007).

As shown in Figure 1-1, the indirect pathway begins with the projections of the
Stl‘iatalpallidal medium spiny neurons onto the external segment of the globus pallidus
(GP e) that releases GABA and the opioid peptide enkephalin (Steiner et al., 1999) and
proj ect onto the subthalamic nucleus (STN). The STN neurons project to the GPi/SNpr
and release glutamate in these brains regions thereby exciting neurons in the Gpi/SNpr

(Squire, 2003). Excitation of the GPi/SNpr neurons that release GABA in the thalamus

The Basal Ganglia

Indirect
Pathway

 

Figure 1-1. Anatomy of the basal ganglia. Neuronal axon terminals are represented as
glutarnatergic (A) and GABAergic (-L). The basal ganglia can alter the activity of the
#1318ng output to the motor cortex using the direct (excitatory) pathway or the indirect
(mhi bitory) pathway on the thalamus. NSDA neurons originating in the substantia nigra
pars compacta (SNpc) terminate in the striatum and regulate the activity of the direct and
Indirect pathways. Cell bodies of the different neuronal populations making up the basal
gangl ia originate or axonal terminals terminate in the cortex, striatum, internal segment of
the globus pallidus (GPi), external segment of the globus pallidus (GPe), subthalamic
311916118 (STN), substantia nigral pars reticulata (SNpr), or thalamus. D1 and D2

Opatnine receptors are located on cell bodies of the medium spiny neurons originating in
the Stl‘iaturn and terminating in the GPi/SNpr or the GPe.

causes inhibition of motor output thereby counteracting the excitatory effects of the direct
pathway in the thalamus (Benarroch, 2007).
The ultimate regulatory mechanism of these two pathways is the NSDA neurons

that synapse on both striatonigral and striatopallidal medium spiny neurons in the

stri atum. The release of DA onto these two populations of medium spiny neurons
regulates the fimction of both the direct and indirect pathways differentially (DeLong and
Wichmann, 2007). Activation of dopaminergic D1 receptors in the direct pathway leads
to exoitation of striatonigral neurons and thereby facilitates motor output by the thalamus.
DA activation of the dopaminergic D2 receptors on the striatopallidal neurons inhibits

these neurons also facilitating motor output (Squire, 2003; Benarroch, 2007)-

The Basal Ganglia in PD

Degeneration of the NSDA neurons within the basal ganglia leads to dysfunction
of the basal ganglia and over-activation of the indirect pathway and an overall inhibition
of motor facilitation by the thalamus (DeLong and Wichmann, 2007). This process
OCCIJrs through the loss of dopaminergic inhibition on striatopallidal GABAergic neurons,
which (in turn) leads to inhibition of the GABAergic GPe neurons. Inhibition of the GPe
leads to firing of the glutamatergic STN neurons onto the GPi/SNpr and inhibition of the
rmotor output by the thalamus (Brotchie, 2003; DeLong and Wichmann, 2007). Overall
inI‘Libition of motor output by thalamus is responsible for the slowness of movement

which occurs in PD patients.

1). DA Synthesis, Release, and Metabolism

DA is a catecholamine which is made up of a benzene ring, an amine group, and
two hydroxyl groups. The synthesis of DA occurs when the dietary amino acid L-
tyrosine is transported into the DA neuron via the large neutral amino acid transporter
(Squire, 2003). L-tyrosine is converted to 3,4-dihydroxyphenylalanine (DOPA) by the
enzy'me tyrosine hydroxylase (TH) via the addition of a hydroxyl group to the benzene
ring of tyrosine (Dunkley et al., 2004). TH synthesis of DOPA is considered the rate
limiting step in DA synthesis because brain levels of L-tyrosine are high enough that TH
is Saturated and not limited by substrate availability. The synthesis of DA is completed
when DOPA is rapidly decarboxylated by the enzyme DOPA decarboxylase (DDC) to
form DA, which is primarily sequestered in synaptic vesicles to be released on demand
(Scrum, 2003).

DA also serves as the precursor for the neurotransmitters norepinephrine and
epinephrine in other catecholaminergic neurons. The formation of norepinephrine occurs
Via the addition of a hydroxyl group to one of the carbon atoms of the amine group on
DA by the enzyme DA B-hydroxylase. Norepinephrine serves as the substrate for the
SYIlthesis of epinephrine by the enzyme phenylethanolarnine N-methyltransferase
(Sqmre, 2003).

TH is the key rate limiting enzyme in the synthesis of DA and its activity can be
regulated in dopaminergic neurons by various mechanisms. TH has 4 serine
phosphorylation sites critical for enzyme activation including serine 8, 19, 31, and 40.
Pllosphorylation of these serine sites is important for regulation of TH activity, though

TH serine 40 phosphorylation is key to maximal activation of the enzyme.

 

Phosphorylation of TH at the various serine residues occurs via kinases such as protein
kinase A and protein kinase C, both of which phosphorylate serine 4O (Dunkley et al.,
2004; Bobrovskaya et al., 2007). Activated TH uses the cofactor tetrahydrobiopterin
(BI—I4), oxygen (02), and Fe“2 to hydroxylate L-tyrosine. In this reaction the Fe2+
component of TH binds L-tyrosine and in the presence of BH4 and Oz, L-tyrosine is
hydroxylated to DOPA, BH4 is converted to dihydrobiopterin (3H2), and one molecule of
H20 is produced (Figure 1-2) (Fitzpatrick, 2003). Regulation of TH involves end-
Pl'Oduct (DA) feedback inhibition, where DA competition with BH4 for the Fe2+ site on
TH reduces TH activity. The availability of BH4 (either limited or in excess) can reduce
TH activity as does limitations in availability of the substrate L-tyrosine (Dunkley et al.,
2004). However, the extent and threshold for initiation of end product inhibition by DA
on TH activity depends on the dopaminergic neuronal population (Figure 1-3).
Following its release into the synapse, DA binds to either pre- or post-synaptic
DA receptors or is taken back up into the DA neuronal terminal via the DA transporter
(DAT) (Storch et al., 2004). Following reuptake by DAT, DA is either re-sequestered
into synaptic vesicles via the vesicular monoamine transporter 2 (VMAT2) or is
metabolized within the mitochondria by monoamine oxidase B (MAO B) to 3,4-
dihBldroxyphenylacetaldehyde (DOPAL), and then to the metabolite 3,4-
dihydroxyphenylacetic acid (DOPAC) via aldehyde dehydrogenase (AD) (Figure 1-3).
DOPAC diffuses out of the neuron and is converted to homovanillic acid (HVA) by
Catechol-O-methyltransferase (COMT) in glial cells. Alternatively DA is converted via

CQMT to 3-methoxytyramine (3-MT) in glial cells, 3-MT is converted to HVA aldehyde

10

(HVAA) by MAO, and HVAA is metabolized to HVA via AD (Figure 1-4) (Squire,

2 003; Galvin, 2006).

11

 

Tyrosine Hydroxylation

3H, 3H,

V Ho
”0% — “0%
002 C02
NH3 / F91” \ NH3

L-Tyrosine 02 I H20 DOP A

TH

FigIJre 1-2. TH synthesis of DOPA from dietary tyrosine. DOPA is synthesized fiom L-
tyro sine by oxidation of L-tyrosine in the presence of tetrahydrobiopterin (BI-I4) and Oz
by TH. Figure is adapted from (Fitzpatrick, 2003).

12

DO PAC

t e

l
DOPACQOCI‘IOWDA— DAT __

AD DOPAL MAOB

TH DDC
Tyrosine —— DOPA—DA-fi VMAT r——’DA

TQAE \j D2 R
Tyrosine

F1 gure 1-3. DA synthesis, release, reuptake, and metabolism at the NSDA axon terminal.
DA is synthesized by hydroxylation of L-tyrosine by TH to form DOPA which is

(130 arboxylated by DDC to form DA. DA is sequestered into synaptic vesicles by uptake
by VMAT. DA is released with the arrival of an action potential into the synapse, taken
up by DAT, and either metabolized by MAO B and AD to DOPAC or re-sequestered into
Synaptic vesicles for re-release by VMAT.

 

 

 

 

 

 

 

 

13

MAO AD
DA — DOPAL—> DOPAC .

COMT COMT

 

 

' MAO AD I
3-MT——> HVAA —-> HVA

Figure 1-4. DA is metabolized by one of two pathways to HVA. DA is converted
from DA to DOPAL to DOPAC to HVA or to 3-MT to HVAA to HVA. Figure is
adapted from (Galvin, 2006).

14

E. DA Neuronal Populations
Dopaminergic neurons are divided into one of ten different neuronal populations

that constitute part of the catecholamine neuronal system within the brain made up of 17
catecholaminergic neuronal groups classified in a caudal to rostral numerical system (A1-
A17) (Figure 1-5). The A1-A7 groups are noradrenergic neurons, whereas the A8-Al 7
groups are dopaminergic neurons. The mesencephalic dopaminergic neurons consist of
the A8 (retrorubal), A9 (nigrostriatal), and A10 (ventral tegmental) populations that are
found within the midbrain. The diencephalic dopaminergic neurons include All
(diencephalspinal), A12 (tuberoinfindibular), A13 (incertohypothalamic), A14
(periventricular-hypophysial/periventricular), and A15 (ventral lateral hypothalamic)
neurons (Bjorklund, 1984a; Lookingland, 2005; Pappas et al., 2008). Two other
dOpaminergic neuronal populations the A16 and A17 are found within the glomerular

layer of the olfactory bulb and the retina, respectively (Hida et al., 1999; Chen et al.,

2000). The function and regulation of the nigrostriatal, ventral tegmental, and

tuberoinfindibular systems have been well characterized (Moore and Wuerthele, 1979;
ROth, 1984; Lookingland et al., 1987b; Lookingland et al., 1987a; Murrin and Roth,
1987; Eaton et al., 1992; Eaton et al., 1994; Lookingland, 2005).

The A9 nigrostriatal dopaminergic (N SDA) neurons originate in the SNpc, project
their axons via the medial forebrain bundle, and primarily terminate in the striatum
(caudate-putarnen) of the forebrain. The A8 dopaminergic neurons originating in the
VentI‘al tegrnental area (VTA) project through the medial forebrain bundle to both the
nucleus accumbens and prefrontal cortex via the mesolimbic DA (MLDA) and

Ines0cortical DA pathways (Fallon and Moore, 1978; Bjorklund, 1984a). The A12

15

tuberoinfindibular dopaminergic (TIDA) neurons originate in the arcuate nucleus of the

hypothalamus and terminate in the median eminence (Moore and Wuerthele, 1979)

16

Air/rum

Willi/lira
ea— Norepinephrine—~—

 

A9

 

 

 

     

"-3

'
V:o."..:o

 

l

  
 

    

Figul‘e 1-5. Catecholaminergic neuronal populations in the rodent brain. Al-A7
populations are noradrenergic and A8-A16 are dopaminergic. Catecholamine neuronal
populations and are located in numerical order in a caudal to rostral direction (Moore,

1987).

17

 

F. DA Receptors
DA receptors are found throughout the brain and five different subtypes of DA

receptorshave been cloned and characterized. All DA receptors are G protein linked
primarily to either G inhibitory (G) or G stimulatory (G,). The receptors have been
classified into two families Dl-like and D2—like (Sealfon and Olanow, 2000).
These two families of DA receptors were first characterized by their functions and ligand
binding properties. The family of D1 receptors (D1 and D5) were determined to
stimulate adenylate cyclase and D2 the receptors (D2, D3, & D4) inhibited adenylate
cyclase (Onali et al., 1985; Missale et al., 1998). These classifications are still
maintained today and have since been well characterized as described.

The D1 receptor is a G5 linked protein which stimulates adenylate cyclase and was
the first of its family to be cloned The D5 receptor was cloned and found to have similar
Properties to the D1 and thus was grouped in the D1 like family (Sunahara et al., 1991).
D1 receptor localization was initially determined by ligand binding studies (Dubois et al.,

1986; Savasta et al., 1986), but since D1 receptor mRN A and protein within the brain has
been determined. D1 receptors have the highest expression level within the brain, and are
maiIlly found within the striatum, nucleus accumbens, olfactory tubercle, limbic system,
hypothalamus, and thalamus (Missale et al., 1998; Sealfon and Olanow, 2000). D5
receptor expression is very limited in the brain and is exclusively found on neurons
rnail’lly in the limbic regions, olfactory tubercle, hippocarnpus, and marmnillary nucleus

(Suhahara et al., 1991; Missale et al., 1998; Sealfon and Olanow, 2000).

18

The D2 receptor is G, linked and inhibits adenylate cyclase. Similar to the D1
family receptor localization studies, initially location of the D2 family of receptors was
determined using ligand binding studies (Onali et al., 1985; Camus et al., 1986).
However, these findings were later confirmed or expanded using mRNA and protein

expression studies within the brain. The D2 receptor is expressed widely throughout the

brain including the basal ganglia, pituitary, striatum, olfactory tubercle, nucleus

accumbens, hypothalamus, VTA, and SNpc (Bunzow et al., 1988; Sesack et al., 1994;

Missale et al., 1998; Sealfon and Olanow, 2000). D3 receptor expression is mainly found

within the nucleus accumbens, olfactory tubercle, hypothalamus, and limbic areas of the
brain, with low expression in the striatum, VTA, and substantia nigra (Sokoloff et al.,
1990; Sealfon and Olanow, 2000). D4 receptors are mainly found in the frontal cortex,
amygdala, olfactory bulb, and hypothalamus (Sealfon and Olanow, 2000).

The D3 and D4 receptors were later cloned and found to have similar properties to
the D2 receptor and were classified in the D2-like family of DA receptors (Bunzow et al.,
1988 ; Sokoloff et al., 1990; Van Tol et al., 1991). One of the main differences between
the D 1 and D2 families of receptors is that the D1 family does not have any introns and

only comes in one form. However, the D2 family contain at least one intron thus
alloWing more then one splice variant to exist for each receptor (Sesack et al., 1994;
Missale et al., 1998).

The role of D1 and D2 receptors in the basal ganglia is important for the
regulation of locomotion and (in the case of D2 receptors) regulation of dopaminergic
neur011a] activity (Missale et al., 1998). D1 receptors are located on GABAergic medium

Spiny neurons within the striatum that project via the striatonigral pathway to the SNpr

l9

and the GPi. These GABAergic medium spiny neurons express substance P and
dynorphin, and D1 receptor stimulation increases the expression of these neuropeptides in
these neurons. D2 receptors are also located on GABAergic medium spiny neurons
within the striatum that project via the striatopallidal pathway to the GPe and release the
neuropeptide enkephalin (Ariano et al., 1992; Yung et al., 1995; Missale et al., 1998).

Stimulation of D2 receptors inhibits the expression of the enkephalin precursor peptide

preproenkephalin in striatopallidal neurons (Missale et al., 1998).

D2 receptors are also on the cell bodies, dendrites, and axon terminals of NSDA
and MLDA neurons, and these “D2 autoreceptors” regulate the synthesis and release of
DA (Roth, 1984; Bozzi and Borrelli, 2006). D2 receptors are also expressed by anterior

pituitary lactotrophs. DA released from TIDA neurons terminating in the median

eminence binds to these receptors and inhibits the release of prolactin from these

lactotrophs (Borgundvaag and George, 1985; Moore, 1987).

20

G. Regulation of DA Synthesis in Central Dopaminergic Neurons
There are three primary regulatory mechanisms of DA synthesis in central

dop aminergic neurons including post-synaptic long loop feedback, pre-synaptic short
loop feedback, and end product inhibition (Moore and Wuerthele, 1979; Roth, 1984;
Ogren et al., 1986). Post-synaptic long loop feedback involves DA action on post-
synaptic DA receptors that inhibit the release of DA from the pre-synaptic terminal via
afferent neuronal inhibition of dopaminergic neuronal activity. Short loop feedback
involves DA in the synapse binding pre-synaptic D2 autoreceptors leading to direct
inhibition of TH activity and the release of DA from the pre-synaptic terminal. End-
product inhibition occurs within DA neurons when the concentration of intracellular DA

suppresses TH activity.

Regulation of dopaminergic neurons can vary amongst groups and these neurons
may be regulated by one or more of these mechanisms. NSDA neurons possess all three
mechanisms of feedback inhibition (Figure 1-6). DA binding to D2 receptors on post-
Synalptic medium spiny neurons leads to post-synaptic long loop feedback inhibition of
N SDA neuronal activity (Ogren et al., 1986; Eaton et al., 1992). D2 autoreceptors on the
axonal terminals of NSDA neurons inhibit TH activity within the cell and the release of
DA into the synapse (Walters and Roth, 1976; Bannon et al., 1981; Foreman et al., 1989;

EatOn et al., 1994; Pappas et al., 2008). End product inhibition occurs when cytoplasmic
ConCentrations of DA within NSDA neurons increases due to DA synthesis or reuptake of
releElsed DA by DAT. Non-sequestered DA can directly inhibit the activity of TH by
competitively inhibiting the binding of tyrosine to the F e2+ site on TH (Demarest and

Mere, 1979).

21

MLDA neurons also possess all 3 mechanisms of DA neuronal regulation,
although MLDA neuronal D2 autoreceptor function is more sensitive than NSDA
neurons (Demarest and Moore, 1979; Demarest et al., 1983; Ogren et al., 1986; Eaton et
al., 1 992). In contrast to NSDA and MLDA neurons, TIDA neurons exhibit only end-

product inhibition and lack both short and long loop feedback mechanisms (Demarest and

Moore, 1979; Berry and Gudelsky, 1991; Pappas et al., 2008).

22

NSDA Neuron

  

 

 

Fi gure 1-6. Feedback regulation of NSDA neuronal activity. Feedback mechanisms
lfle l ude post-synaptic long loop feedback, pre-synaptic short loop feedback (red dashed
hue), and end product inhibition (blue dashed line).

23

H. Indices of DA Neuronal Integrity and Activity
Several indices are commonly used to determine the integrity of dopamine
neurons. The integrity of dopaminergic terminals can be determined by measuring the
concentrations of DA and/or protein content of TH and DAT in the dopaminergic axon
terminal region. DA concentrations are primarily located in synaptic storage vesicles in
ax on terminals and are measured using high performance liquid chromatography with
e1 ectrochemical detection (HPLC-EC). A decrease in synaptic vesicular and cytoplasmic
DA concentrations reflects a loss in dopaminergic terminal integrity (Drolet et al., 2004).
The protein content of cytoplasmic TH and membrane-bound DAT found in the striatum
are measured using Western blotting and have been used as an index of terminal integrity
(Ti llerson et al., 2002; J akowec et al., 2004). A decrease in either TH or DAT protein
re f1 ects a loss of dopaminergic neuronal terminals, especially DAT which is almost
ex e lusively expressed by dopaminergic neurons.

Cell body integrity of dopaminergic neurons is determined by counting the
n‘-—111'1ber of TH immunoreactive cells in regions of the brain containing cell bodies of
tll'EBSe neurons (J ackson-Lewis et al., 1995; Petroske et al., 2001). However, if a change
in TH cell body number is found in a region, it is critical to count cells in this region
using another neuronal cell marker that labels all neuronal cell bodies such as

Ni Ssl/Cresyl Violet. By doing this and comparing the number of TH immunoreactive
Ce] 1 s to the number of Nissl positive cells one can discern if there is clearly a reduction in
Ce 1 1 number or if there is a decrease in the number of cells expressing TH (J ackson-Lewis

er; al., 1995). The amount of TH protein determined by Western blotting in cell body

24

regions of the dopaminergic neurons is also used as an indicator of TH cell body
integrity.
The ratio of either of the two major DA metabolites DOPAC or HVA to DA itself
(DOPAC/DA or HVA/DA) is used as an index of DA neuronal activity (Moore and
Wuerthele, 1979). Activity of the DA neuron consists of synthesis, release, reuptake, and
m etabolism of DA. Increased activity is indicated by an increased ratio due to a decrease
in DA stores and/or an increase in metabolism of DA to DOPAC or HV A due to greater
rel ease and reuptake of DA. A decrease in either of the ratios would be reflective of a
red notion in dopaminergic neuronal activity (Moore and Wuerthele, 1979; Lookingland,

2005).

25

I. Serotonin Synthesis and Neuronal Function

The dorsal and median raphe nuclei is the location of cell bodies for the neurons

which project to the forebrain and release the neurotransmitter serotonin (5-
.hydroxytryptamine; 5HT), another biogenic amine neurotransmitter. Serotoninergic
n eurons project axons via ascending and descending pathways throughout the brain, but
mainly in a rostral direction. Specific regions that 5HT neurons terminate include the

striatum, nucleus accumbens, cerebral cortex, substantia nigra, hypothalamus and spinal

cord (Moore et al., 1978; Bjorklund, 1984b).

5HT is an indolarnine synthesized from the amino acid L-tryptophan, which (like
tyrosine) is taken up by the large neutral amino acid transporter. Synthesis of 5HT is
sub strate limited by the low concentrations of dietary L-tryptophan (Wurtman and
Fenlstrom, 1975; Squire, 2003). L-tryptophan is hydroxylated by the rate limiting
enz yme tryptophan hydroxylase to L-S-hydroxytryptophan, and decarboxylated by
31‘ Ornatic-L-amino acid decarboxylase to 5HT. 5HT is taken up into synaptic vesicles by
VMATZ and stored until release (Squire, 2003). Following its release 5HT binds to pre-
and post-synaptic 5HT receptors (Cerrito and Raiteri, 1979; Fink and Gothert, 2007), and
is inactivated by re-uptake into pre-synaptic terminals by the serotonin reuptake
tr aJ‘lsporter (SERT). 5HT can be metabolized within the serotoninergic neuron by MAO
to 5 ~hydroxyindole acid aldehyde and oxidized to 5-hydroxyindole acetic acid (SHIAA)

(Squire, 2003).

Similar to dopaminergic neurons, the integrity of serotoninergic neurons can be

determined by measuring the concentrations of 5HT within brain regions containing

Rona] terminals of serotoninergic neurons, including the striatum or nucleus accumbens.

26

The activity of serotoninergic neurons can also be estimated like dopaminergic neurons
by determining the ratio of the 5HT metabolite SHIAA to 5HT (SHIAA/SHT) ratio (Tian

et al., 1992; Darmani et al., 2003).

27

 

J. Neurotoxic Models of PD

MPT P

MPTP is a relatively selective neurotoxin for dOpaminergic neurons. MPTP was
discovered to cause a Parkinson’s like movement disorder in 1982 when a group of
heroin users in California accidentally injected themselves with the neurotoxin that was a
b y—product of an attempted synthesis of a meperidine analog (Langston et al., 1983). The
patients presented soon after with a movement disorder resembling PD. Later it was
determined that MPTP was the offending agent following analysis of samples of the
drugs the patients injected. The use of MPTP as a potential model of PD followed the

i ncidental finding of Parkinson’s like symptoms in these drug users (Dauer and

Przedborski, 2003).
MPTP is a lipophilic compound that readily crosses the blood brain barrier.

MPTP is converted to MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridine) primarily in
g1 ial cells by the enzyme MAO B and is oxidized to its active metabolite MPP+ (1-
methylA-phenylpyridinium) (Markey et al., 1984; Dauer and Przedborski, 2003). MPP+
has a high affinity for DAT and is selectively transported by DAT into dopaminergic
lieurons (Figures 1-7 & 1-8) (Javitch et al., 1985 ; Gainetdinov et al., 1997; Dauer and
PI‘Zedborski, 2003). Once inside the DA neuron, MPP+ blocks Complex I of the
mitochondrial respiratory chain thereby inhibiting the synthesis of ATP causing energy
Clepletion and oxidative stress (V yas et al., 1986). MPP+ is also sequestered into synaptic
Vesicles containing DA via uptake in to vesicles by the VMAT2 (Liu et al., 1992).

Q)(idative stress also occurs by MPP+ oxidation of DA forming DA quinones which are a

1“fee radical species of DA that induce further production of other free radical species and

28

 

can modify proteins altering neuronal function (Figure 1-8) (Klaidman et al., 1993; Dauer

and Przedborski, 2003; Lee et al., 2003). The method of cell death attributed to MPTP

induced neurotoxicity is dependent on the dosing paradigm and frequency of

administration but apoptosis (programmed cell death), autophagy, and necrosis have all

been implicated (Zhu et al., 2007).
MPTP is likely the most common model used to study PD. MPTP can elicit its

neurotoxic effects not only in humans but also in monkeys and mice (Burns et al., 1983;
H eikkila et al., 1984). The differences in MPTP dosage, dosing fi'equency, interval
1) etween injections, and time point following MPTP treatment when the animals are

ki lled can produce differential effects on dopaminergic terminal degeneration (Sundstrom

et al., 1987; Sundstrom et al., 1990; Behrouz etal., 2007).

29

      
    
 
   

Blood Brain
Barrier

Glial Cell

Dopamine .
Neuron A

Pi gure 1-7. Metabolism of MPTP to MPP+ and transport of MPP+ into DA neurons.

PTP crosses the blood brain barrier as a lipophilic compound and is metabolized by

MAOB, than oxidized to its active metabolite MPP+ and released via an unknown
IIlechanism by glial cells to be taken up into the dopaminergic neuron. Figure is taken
fi‘Om (Dauer and Przedborski, 2003).

30

 

 

MPP+ blocks
the electron

    
  
 

 

 

 

 

 

 

. transport chain
Synaptic
MPP Vesrcle Mitochondria
+ VMAT
'\

MPP+ MPi=+

     

 

DAT

t
MPP+

 

 

 

Fi gure 18 Effects of MPP+ at the dopaminergic neuronal terminal. MPP+ is transported
Into the dopaminergic neuronal terminal by DAT and inhibits Complex I in the

mitochondrial respiratory chain or is sequestered into synaptic vesicles by uptake into
Vesicles by VMAT.

31

 

6—Hydr0xydopamine (6-0HDA)

The 6-hydroxydopamine (6-OHDA) model was the first established rodent model
for PD. 6-OHDA is a hydroxylated analogue of DA and is taken up by both DAT and the
norepinephrine transporter (NET) into catecholaminergic neurons (Bove et al., 2005). 6-
OHDA accumulates in the cytosol and upon oxidation induces reactive oxygen species

production and the formation of quinone species within the cell (Heikkila and Cohen,

1 973). Although reactive oxygen species are the primary cause of 6-OHDA mediated
death, the quinone species also contribute by reacting with cellular DNA and proteins
causing cellular damage (J onsson and Sachs, 1975; Bove et al., 2005; Drechsel and Patel,
2008). The death of dopaminergic neurons induced by 6-OHDA can also activate
mi croglial cells within the brain which in turn directly induce the loss of NSDA neurons
(Rodriguez-Pallares et al., 2007).

6-OHDA does not easily cross the blood brain barrier and accordingly needs to be
administered directly into the brain by stereotaxic injection. In the various 6-OHDA
rodent models of PD, the neurotoxin is injected directly into the substantia nigra, medial
f0rebrain bundle or striatum, either unilaterally or bilaterally. Injection into the

Substantia nigra or medial forebrain leads to a more substantial and rapid loss of NSDA

r1elrrons, compared to injection into the striatum which involves a more progressive
retrograde loss of NSDA neurons and axonal terminals over 1-3 weeks (Bove et al.,

2 005). Unilateral injection of 6-OHDA is advantageous in that it selectively lesions one
Side of the NSDA neuronal system causing DA agonist induced turning behavior which is
proportional to the extent of the lesion (Przedborski et al., 1995). This model system is

t1lerefore used to test potential anti-parkinson’s drugs, by quantifying the circling

32

 

 

 

\J‘ W‘fiz‘

behavior of the rodent. Although, 6-OHDA is not completely selective for dopaminergic

neurons, it is still used frequently as a rodent model of PD.

Rotenone
Rotenone is a common pesticide which (like MPTP) is lipophilic and inhibits

Complex I of the mitochondrial respiratory chain (Bove et al., 2005). Rotenone has only
recently been used as a model of PD by a limited number of investigators (Betarbet et al.,
2 000). The primary mechanism of rotenone toxicity is its ability to inhibit Complex I and
induce ATP depletion and ROS formation (Bove et al., 2005; Drechsel and Patel, 2008).
R0 tenone also directly activates microglial cells, which contributes to microglial
mediated induced death of NSDA neurons (Gao et al., 2002a; Sherer et al., 2003). Since
rotenone is the active form of the neurotoxin and is lipophilic, it is not selective for
dopaminergic neurons. Preferential loss of NSDA (as opposed to MLDA) neurons has
been demonstrated following rotenone administration, likely do the higher sensitivity of
NSDA neurons to the deleterious effects of oxidative stress (Drechsel and Patel, 2008).
I{Otenone administration causes protein aggregates within NSDA neurons that are similar
‘30 Lewy bodies, suggesting that this model may be ideal for studying pathology
associated with proteosome and related protein metabolism deficits (Betarbet et al., 2000;
Dauer and Przedborski, 2003). The use of the rotenone model is considered limiting,

l'lowever, due the lack of selectivity and the extent of disruption of NSDA neurons.

33

 

"h

K Inflammation in the Brain

Microglial Cells

Microglial cells are the resident immune cells in the brain that are derived from a
myeloid lineage and are similar to macrophage cells found outside of the central nervous
system (Rock et al., 2004). Microglia are estimated to constitute about 15% of all cells
within the brain, but the number of microglia can vary among brain regions with some
areas having a higher number of microglia than others (Kim et al., 2000; Rock et al.,

2004). Microglial cells serve as a form of immune surveillance in the central nervous
system in their resting state and have a rarnified morphology (Vilhardt, 2005). Activation
of Inicroglial cells leads to a rapid transformation from a ramified shape to an amoeboid
circular shape. Microglial activation also results in a significant increase in the number
0 f cell membrane receptors involved in the inflammatory response and an increase in the

release of inflammatory cytokines and free radical species (Rock et al., 2004; Vilhardt,

2005).

Microglial activation can occur in response to infection, brain injury and neuronal
Cell death, and leads to microglial proliferation and migration, increased phagocytic
activity, and ability to present antigens to other immune cells (V ilhardt, 2005; Liu,
2006). Activated microglia also release pro-inflammatory mediators including tumor
rlecrosis factor alpha (TNF-a), interleukin-1 beta (IL-113), prostaglandins, chemokines,
ROS and reactive nitrogen species (Hanisch, 2002). Activated microglia release the anti-
i rlflarnmatory cytokine IL-10 and neurotrophic factors (Vilhardt, 2005). The ability of

rt‘licroglia to identify infection, injury, or cell death is beneficial in that an immune

reaction is initiated which recruits other microglia via cytokines to remove dead or dying

34

 

cells. The detrimental effects of microglial activation include increased numbers of
activated microglial which prolongs the release of pro-inflammatory mediators and free
radical species. This process termed “microgliosis” continues throughout the cycle of

microglial activation and induces death of surrounding neurons (Block and Hong, 2005).

M icroglial Activation and PD

Inflammation induced neuronal death mediated by activated microglia has been
implicated in the process of neurodegeneration in several diseases including PD,
Alzheimer’s disease, multiple sclerosis, and stroke. PD patients and animal models of
PD demonstrate a greater number of microglia within the substantia nigra region
compared to controls (McGeer et al., 1988; Imamura et al., 2003; Barcia et al., 2004;
Ouchi et al., 2005), making NSDA neurons more likely to experience microgliosis (Kim
et al., 2000). Microglial mediated cell death of NSDA neurons likely occurs in humans
and is seen in several animal models of PD.

Several different neurotoxins and inflammatory mediators are used in animal
models to study PD, and have yielded valuable information concerning potential factors
which lead to the development of the disease. Several of these animal models either
directly or indirectly activate microglia (Figure 1-9). The MPTP and 6-OHDA
rlellrotoxin models of PD indirectly activate microglia by inducing NSDA neuronal cell
1083 (Gao et al., 2003; Block and Hong, 2005). The neurotoxin rotenone induces NSDA
tleIrronal cell death, but also activates microglia directly (Gao et al., 2002a). However,

the lipopolysaccharide (LPS) model of PD allows the exclusive activation of microglia

35

LPS, Rotenone MPTP, Rotenone, 6-0HDA

  
 

Resting Microglia

Healthy Neuron

Activated Microglia Sick Neuron

Self Amplifying Neurotoxicity

Figure 1-9. Direct and indirect activation of microglia induces microgliosis in models of
PD- LPS and rotenone directly activate resting microglial cells and the neurotoxins 6-
OHDA, MPTP, and rotenone indirectly activate microglial cells by inducing neuronal
death leading to microgliosis. Figure taken from (Hald and Lotharius, 2005).

36

.f—

 

without direct induction of NSDA neuronal cell death (Liu et al., 2000a; Iravani et al.,

2005; Roy et al., 2006).

LPS as an Inflammatory Model of PD
LPS is a component of the cell wall of gram negative bacteria which directly

activates resident microglia in the brain via the Toll-like receptor 4 (TLR4) (Lehnardt et

al., 2003; Qin et al., 2005). LPS is able to exclusively induce microglia activation
because neurons lack TLR4 receptors (Lehnardt et al., 2003). This allows study of
m ieroglial mediated loss of NSDA neurons, without concurrently inducing NSDA
neuronal loss by a neurotoxic mechanism. Most studies using LPS in in vivo models of
PD directly inject LPS into the substantia nigra or striatum of rodents (Castano et al.,
1 998; Kim et al., 2000; Hsieh et al., 2002; Iravani et al., 2002; Zhou et al., 2005). This
allows a relatively localized activation of microglia in these regions leading either to loss
of NSDA neuronal cell bodies followed by retrograde loss of axon terminals. In contrast,
LPS can also be injected systemically but induces an inflammatory reaction throughout
tIle brain and does not cause loss of NSDA neurons till at least 7 months (Qin et al.,

2007).

37

 

L. Neurogenesis and Sildenafil

Neurogenesis and stem cell replacement therapy have been proposed as ways to
replace NSDA neurons lost in PD. Stimulation of endogenous neural stem cells within
the brain would be most ideal as this would prevent a host-immune response to donor
stem cells (Borlongan et al., 1996). One potential mechanism to induce neurogenesis is
through inhibition of the enzyme phosphodiesterease 5 (PDES). PDES catalyzes the

break down of 3’,5’-cyclic guanosine monophosphate (cGMP) to 5’-guanosine
monophosphate (Corbin and Francis, 1999; Kulkami and Patil, 2004). Increased cGMP
levels following inhibition of PDE5 using the drug sildenafil increases neurogenesis of
subventricular derived stem cells (Wang et al., 2005). Sildenafil also increases the
concentrations of cGMP in the brain, promotes neurogenesis from the subventricular
zone within the brain, and improves neurological recovery following ischemic stroke in
I‘Odents (Zhang et al., 2002; Zhang et al., 2005; Zhang et al., 2006a). This information
Suggests that sildenafil may be a potential mechanism to induce neurogenesis in a rodent

rrlOdel of PD. Studies outlined in Chapter 3 of this disseration tested this possibility.

38

 

 

M. The Role of N icotinamide Adenine Dinucleotide Phosphate (N ADPH) Oxidase in
Microglial Mediated Death of NSDA Neurons
The ability of microglia to produce the ROS superoxide in models of PD has been
attributed to the activation of the enzyme NADPH oxidase (Gao et al., 2002a; Gao et al.,
2003). NADPH oxidase is found in several cell types in the central nervous system
including microglia, astrocytes, and neurons (Serrano et al., 2003; Abramov et al., 2005;
Block and Hong, 2005). The activation of microglial NADPH oxidase contributes to the
process of microgliosis which is detrimental to NSDA neurons.

As shown in Figure 1-10, NADPH oxidase is composed of the cytosolic GTPase
protein Racl and subunits in the cytosol (p67Phox, p47Phox, p40Phox) or plasma
membrane (gp91Phox, p22Phox). The cytosolic subunits of NADPH oxidase exist as a
Complex under resting conditions, and the plasma membrane bound subunits exist as a
Complex known as cytochrome b553 (Babior, 1999; Groemping and Rittinger, 2005;
SUIIrimoto et al., 2005). NADPH oxidase is activated when the serine residues on the
cyllosolic subunit p47Phox are phosphorylated inducing a conformational change of the
pretein. This change allows sites that interact with the membrane bound subunit
p22Phox to be accessible, enabling the cytosolic complex to bind to the membrane. The
p67Phox subunit bound to p47Phox and the GTPase protein Racl function together to
aretivate the enzyme. The binding of Racl to p67Phox, leads to the interaction of

p 6‘7Phox with gp91Phox, resulting in superoxide production. The cytosolic subunit
p <l‘OPhox aids in translocation of the cytosolic subunits to the membrane and is involved
in binding the complex to the membrane (Groemping and Rittinger, 2005; Sumimoto et

a1» 2005).

39

Activation of NADPH Oxidase

 

NADPH NADP+

Figure 1-10. NADPH oxidase activation and production of superoxide in microglial
cells. The cytosolic NADPH oxidase subunits p67Phox, p47Phox, and p40Phox
translocate along with the GTPase protein Racl to the membrane bound subunits
gp91Phox and p22Phox inducing the production of superoxide (02’) by the transfer of an
electron from NADPH to molecular oxygen (02).

4O

NADPH induced superoxide or its metabolites hydrogen peroxide and hydroxyl
radical play a large role in microgliosis. Hydrogen peroxide induces the expression of
the protein inducible nitric oxide synthase (iNOS) that synthesizes the reactive nitrogen
species, nitric oxide. Nitric oxide can react with superoxide to produce another reactive
nitrogen species, peroxynitrite (Pawate et al., 2004). Nitric oxide and peroxynitrite both
inhibit the mitochondrial respiratory chain thereby inducing ATP depletion, free radical
species production, and oxidative stress which contributes to the loss of NSDA neurons
(Ebadi and Shanna, 2003; Tieu et al., 2003).

Microglial NADPH oxidase activation also induces the production of pro-
inflammatory cytokines such as TNF-a and lL—l B. TNF-a release recruits other
microglial cells to the site of activation and induces the release of more TNF-a (Kariko et
al., 2004; Block and Hong, 2005; Doyle and O'Neill, 2006). IL] [3 similar to TNF-a can
induce the production of more pro-inflammatory cytokines and activation of microglia
(Basu et al., 2004). Microglial proliferation and release of pro-inflammatory cytokines
leads to the migration of other resident microglia] in the brain that may initially be
beneficial, but the activation of microglial can lead to microgliosis (Block and Hong,

2005).

41

N. Cannabinoids (CB) and Endocannabinoids (ECB)

CB are the psychoactive compounds derived from the plant Cannabis sativa also
known as marijuana. The primary active compound obtained from marijuana is A9-
tetrahydrocannabinol (Ag-THC) a lipophilic compound originally isolated from the plant
in 1964 (Gaoni, 1964). Other psychoactive, but less potent CB compounds were later
isolated. The properties of CB include anti-emetic, anti-inflammatory, and psychoactive
effects raising the possibility of the existence of ECB (Svizenska et al., 2008). The first
ECB, anandamide was discovered in 1992 and others followed including 2-
arachidonoylglycerol (2—AG), noladin ether, and virodhamine (Devane et al., 1992;
Mechoulam etal., 1995; Hanus et al., 2001; Porter et al., 2002).

ECB are lipophilic compounds derived from the lipid precursor arachidonic acid.
The two main ECB anandamide and 2-AG are found within the brain and several other
tissues (Rodriguez de F onseca et al., 2005). ECB are generally produced in an on
demand manner and function in several different biological systems including but not
limited to neuronal, inflammatory, cardiovascular, gastrointestinal, and pain pathways
(Rodriguez de Fonseca et al., 2005; Svizenska et al., 2008).

Anandamide is synthesized by the transfer of arachidonic acid from
phosphatidylcholine within biologic membranes to phosphatidylethanolamine via the
calcium dependent enzyme N-acyltransferase (NAT) forming the anandamide precursor
N -arachidonylphosphatidylethanolamine (NArPE). NAT is regulated by CAMP which

increases enzyme activity via the phosphorylation of protein kinase A (PKA). A NAPE
sp ecifrc phospholipase D then synthesizes anandamide and phosphatidic acid from

NAPE (Di Marzo et al., 1994; Rodriguez de Fonseca et al., 2005; Basavarajappa, 2007).

42

2-AG is synthesized by one of two possible mechanisms where either phospholipase C
(PLC) hydrolyzes membrane phospholipids forming diacylglycerol (DAG) or DAG is
formed by hydrolysis of phosphatidic acid via Mg2+ or Ca2+ dependent phoshpatidic acid
phosphohydrolase activity. DAG is converted to 2-AG by DAG lipase (Bisogrro et al.,
1999; Basavarajappa, 2007).

Inactivation of ECB begins with uptake of anandamide or 2-AG into the cell via
the anandamide membrane transporter (AMT). Following uptake anandamide is
metabolized via hydrolysis by fatty acid amidohydrolase (FAAH) within neurons
(Cravatt et al., 1996; Giang and Cravatt, 1997; Thomas et al., 1997; Egertova et al.,
2003). 2—AG is also taken into neurons via the AMT and is metabolized by
monoacylgycerol lipase (MAGL) to 2-arachidonyl lysophosphatidic acid (Dinh et al.,
2002; Blankman et al., 2007). 2-AG can also be degraded by FAAH like anandamide, or
via enzymatic oxygenation by cyclooxygenase-2 (COX-2) into prostaglandins

(Basavarajappa, 2007).

Localization of CB Receptors within the Central Nervous System

CB receptors are found throughout the central nervous system and are involved in
both neuronal and immune fimction. Within the brain CB activity has mainly been
associated with two receptors, the CB] and CB2 receptors. CB1 receptors are found
widely throughout the brain with especially high concentrations in the basal ganglia
including the striatum, globus pallidus (GP), and substantia nigra pars reticulata (SNpr).

C81 is also expressed in the hippocarnpus, cerebellum, cerebral cortex, hypothalamus,

43

and olfactory bulb (Herkenham et al., 1990; Herkenham et al., 1991b; Hohmann and
Herkenham, 2000; Liu et al., 2003; Wittmann et al., 2007).

CB2 receptors are primarily located on micro glial cells within the central nervous
system and are upregulated by neuronal inflammation and microglial activation (Benito
et al., 2003; Maresz et al., 2005; Mukhopadhyay et al., 2006; Ashton et al., 2007; Nunez
et al., 2008). CB2 mRNA and protein expression have also been found within and on
neurons in the cerebellum and hippocampus (Van Sickle et al., 2005; Gong etal., 2006;
Onaivi et al., 2006). CBZ receptors on one population of neurons plays a role in emesis

regulation, but others have only minimally been characterized (Van Sickle et al., 2005).

Mechanism of Induced Synthesis and Release of E CB within the Brain

ECB are synthesized de novo from lipid precursors and are not stored in synaptic
vesicles like typical neurotransmitters. ECB involved in neuronal regulation are typically
released from post-synaptic neurons and act on CB1 receptors on the pre-synaptic
terminals of inhibitory gamma-aminobutyric acid (GABA) and excitatory glutamate
neurons (Mackie, 2008). C31 and CB2 receptors are G protein linked receptors which
inhibit adenylate cyclase thereby reducing production of the second messenger CAMP.
CBl receptors are also linked to G8 proteins (Demuth and Molleman, 2006).

The type of pre-synaptic cell, method of ECB production, and post-synaptic cell
receptor type determines the terminology of the specific ECB mediated feedback
inhibition occurring at a synapse. The predominate form of CB inhibition is
depolarization induced suppression of neurotransmission involving a transient

suppression of GABA or glutamate release from the pre-synaptic cell (Mackie, 2008).

44

GABA or glutamate induced depolarization of the post-synaptic cell causes an increase in
intracellular dendritic Ca2+ concentrations which triggers the production of ECB (Diana
and Marty, 2004; Mackie, 2008). ECB are released retrogradely and inhibit Ca2+
channels on the pre-synaptic terminal via CB1 receptors and inhibit the release of GABA
or glutamate (Figure 1-11) (Kreitzer and Regehr, 2001; Wilson et al., 2001; Wilson and
Nicol], 2001).

A second method of ECB feedback inhibition is metabotropic induced
suppression of neurotransmission which occurs when post-synaptic metabotropic
glutamate receptors activate phopholipase CB and diacylglycerol lipase, thereby
triggering the formation of ECB from phospholipids of the membrane. ECB retrogradely
feedback on pre-synaptic CB1 receptors reducing GABA or glutamate release. This
process requires little calcium (Ca2+) in the post-synaptic cell (Mackie, 2008).

A third form of ECB feedback is long term depression where prolonged low
frequency stimulation of glutamatergic postsynaptic cell metabotropic receptors causes
high levels of ECB production. This results in prolonged CB1 receptor activation leading

to long term inhibition of glutamate release (Robbe et al., 2002). One other mechanism is

45

Pre-synaptic Cell

  
 

CB Receptor

Post-synaptic Cell

Figure 1-1 1. Depolarization induced suppression of neurotransrrrission by cannabinoids.
Release of neurotransmitter (NT) from a pre-synaptic cell triggers Ca2+ entry into the
post-synaptic cell leading to ECB synthesis from lipid precursors. ECB are then released
from the post-synaptic cell to inhibit release of NT from the pre-synaptic terminal via
action at the CB1 receptor.

46

ECB inhibition of neuronal excitation where repeated depolarization of a neuron triggers
increased intracellular Ca2+ concentration inducing ECB production. ECB are released
and act on CBl receptors on the same cell activating inwardly rectifying K+ channels

causing hyperpolarization of the neuron (Mackie, 2008).

Role of ECB in the Basal Ganglia

NSDA neurons are part of the basal ganglia regulating motor output from the
thalamus. Although NSDA neurons do not express CB1 or CB2 receptors the complex
network of the basal ganglia is highly regulated by ECB at both glutamatergic and
GABAergic synapses via CBl receptors. The striatum contains glutamatergic terminals
projecting from the motor cortex which possess pre-synaptic CB1 receptors that regulate
the release of glutamate by ECB feedback from medium spiny neurons within the
striatum (Julian et al., 2003; Matyas et al., 2006; Narushima et al., 2006; Matyas et al.,
2008). GABA release from medium spiny neurons in the striatum is also regulated by
post-synaptic feedback of CB from the GPe, GPi, and SNpr. The STN making up part of
the indirect pathway of the basal ganglia projects glutamatergic neurons to the GPi and
SNpr which have CB1 receptors and are regulated by ECB release within the GPi and
SNpr (Figure 1-12) (Benarroch, 2007). The many parts of the basal ganglia which
possess CB1 receptors indicates the important role ECB function can play in NSDA

neuronal regulation.

47

Cannabinoid Receptor
Expression in the Basal Ganglia

      
 

    

Direct
Pathway

Figure 1-12. CB1 receptor expression within the basal ganglia. CB1 receptors are
located on both glutamatergic (A) and GABAergic (-L) terminals within the basal ganglia.
CB1 receptors are on cortical neurons terminating in the striatum. Medium spiny neurons
projecting to the globus pallidus interna (GPi), substantia nigra pars reticulata (SNpr),
and globus pallidus extema (GPe) have axonal terminal CBl receptors. Glutamatergic
neurons originating in the subthalamic nucleus (STN) and terminating in the GPi and
SNpr also have terminal CB1 receptors. ECB action at different locations within the
basal ganglia can alter the activity of the direct (excitatory) pathway or the indirect
(inhibitory) pathway on the thalamus, and also indirectly effect NSDA neurons
originating in the substantia nigra pars compacta (SNpc) and terminating in the striatum.

48

CB Receptors and Microglial Function

Microglial cells express both CB1 and CB2 receptors (Waksman et al., 1999;
Franklin and Stella, 2003). CB1 and CB2 receptors on microglial cells primarily mediate
anti-inflammatory firnctions upon stimulation, although recently they were also suggested
to induce inflammation (Roche et al., 2008).

CB1 receptors on microglial cells promote the anti-inflammatory effects of CB by
reducing the expression of the iNOS protein. A reduced content of iNOS leads to a
reduction in the production of nitric oxide by microglia (Waksman et al., 1999). This
effectively reduces the production of peroxynitrite potentially limiting microglial
mediated loss of NSDA neurons.

CB2 receptors are responsible for most of the anti-inflammatory effects of CB in
the brain. Micro glial activation increases CB2 receptor expression in vitro and in several
neuroinflammatory diseases or conditions such as multiple sclerosis, hypoxic stroke,
Alzheimer’s disease, and amyotrophic lateral sclerosis (Benito et al., 2003; Maresz et al.,
2005 ; Mukhopadhyay et al., 2006; Yiangou et al., 2006; Ashton et al., 2007). CB2
agonists act as anti-inflammatory agents by reducing the production of TNF-a and nitric
oxide, but induce microglial cell migration and proliferation (Franklin and Stella, 2003;
Walter et al., 2003; Carrier et al., 2004; Ehrhart et al., 2005; Eljaschewitsch et al., 2006;
F emandez-Lopez et al., 2006). Microglia produce ECB that may act in an autoregulatory
fashion on CBZ receptors on microglial cells thereby feeding back to reduce the
production of inflammatory mediators (Walter et al., 2003; Carrier et al., 2004). CB2
agonist use in neuropathological models involving significant activation of micro glia,

leads to an attenuation of microglial activation and slows the progression of disease

49

demonstrating the anti-inflammatory effects of CB acting at the CB2 receptor (Ramirez et
al., 2005; Kim et al., 2006).

The pro-inflammatory effects of CB have recently been suggested, whereby
elevation of ECB led to increased TNF-a plasma concentrations. TNF-a which induces
activation and migration of microglia and the production of pro-inflammatory cytokines
is therefore detrimental. These pro-inflammatory effects of ECB are reversed in the
presence of CB1 or CBZ antagonist (Roche et al., 2008). These differential findings

make the role of CB in microglial function controversial.

50

O. Dissertation Objectives

Several mechanisms or pharmacological agents to slow or halt the progression of
PD have been evaluated. Most of these potential treatments were aimed at reducing
inflammation, oxidative stress, mitochondrial dysfimction, or protein aggregation in an
attempt to maintain the integrity of remaining NSDA neurons. Alternatively,
administration of neurotrophic factors, implantation of stem cells, or stimulation of adult
stem cells already residing in the brain have been attempted to induce the production of
new dopaminergic progenitor cells.

Three potential mechanisms of neuroprotection are tested in this dissertation, which are:

Specific Aim #1: Determine if sildenafil induces neurogenesis of NSDA neurons
following chronic MPTP treatment by increasing cGMP concentrations in the brain.
Hypothesis: Sildenafil will attenuate the MPTP induced loss of NSDA neurons
when treatment is initiated before, during, or after the completion of chronic
MPTP treatment.
Specific Aim #2: Determine the pharmacokinetics of apocynin in the brain and examine
if apocynin is neuroprotective in an MPTP model of PD by reducing activation of
NADPH oxidase.
livpothesis #1: Apocynin rapidly crosses the blood brain barrier following
systemic administration.
Hypothesis #2: MPTP will induce activation of NADPH oxidase that will
contribute to the loss of NSDA neuronal terminals, and thus be attenuated with

apocynin treatment.

51

Specific Aim #3: Determine if mice lacking CB1 and CB2 receptors have normal

dopaminergic and serotoninergic neuronal integrity, activity, and/or regulation.
Hypothesis #1: A lack of functional CB1 and CBZ receptors will alter the
integrity of NSDA, MLDA, TIDA, or serotoninergic neurons.

Hypothesis #2: The activity and/or regulation of dopaminergic and serotoninergic

 

neurons will be altered in mice lacking functional CB1 and CBZ receptors.
Specific Aim #4: Determine the effects of CB1 and CBZ receptor mutation deletion and
pharmacological blockade on NSDA neuronal integrity and activity following acute or
sub-chronic MPTP or peripheral LPS treatment.

Hypothesis #1: Lack of CB1 and CB2 receptors will increase MPTP induced loss

 

of NSDA axon terminals and cell bodies, and compensatory activation of these

neurons.

Hypothesis #2: Acute peripheral LPS treatment of WT and CBl/CB2 KO mice

 

will alter dopaminergic neuronal integrity or neuronal activity.

Each of these specific aims is investigated and hypotheses tested in this
dissertation by examining if NSDA neuronal integrity, activity, or microglial activation is
altered with either pharmacological treatment or genetic knockout of CB1 and CB2

receptors within the mouse models of PD.

52

Chapter 2: Materials and Methods

A. Animals
Animals

Male C57BL/6 mice (Jackson Labs, Bar Harbor, ME) age 8-10 weeks were used
in most experiments, and as Wildtype (WT) controls (CB1 (+/+), CB2 (+/+)) for all
studies using mice lacking both CB1 and CB2 receptors (CB1 (-/-), CBZ (-/-)) on a
C57BL/6 background. CB1 (-/-)/CB2 (-/-) (CB1/C82 knockout (KO)) mice were
obtained fi'om Drs. Norbert Kaminski and Barbara Kaplan who maintain a breeding
colony of CBl/CBZ KO mice at Michigan State University. Mice used for some
preliminary pharmacology experiments were obtained from a laboratory colony at
Michigan State University maintained by Drs. John Goudreau and Keith Lookingland and

are derived from a cross of the B6-129X and C57BL/6 mouse strains.

CBI/CBZ Receptor K0 Mice

The sequence for the CBI receptor in the mouse is located within one exon on
chromosome 4 (Zimmer et al., 1999). CB1 receptor KO mice were previously created by
replacing most of the CB1 receptor coding sequence between amino acids 32 and 448
with a phosphoglycerate kinase (PGK)-neomycin (neo') cassette in MP12 embryonic stem
cells using homologous recombination (Zimmer et al., 1999). These stem cells were
used to create chimeric mice which were bred with C57BL/6 mice to create heterozygote
CB1 receptor (+/-) mice. Heterozygote CB1 (+/—) mice, were bred to create CB1 receptor

(-/-) KO mice (J arai et al., 1999; Zimmer et al., 1999; Gerald et al., 2006).

53

The CB2 receptor DNA encoding sequence is also contained within a single exon
on chromosome 4. The CB2 gene was inactivated (Buckley et al., 2000) by insertion of a
PGK- neor cassette into the gene replacing 341 base pairs in the 3' coding exon of the
CBZ receptor gene using homologous recombination in the embryonic stem cell line 129.
Chimeric mice were generated with embryonic stem cells and crossed with C57BL/6
mice to create heterozygote CB2 (+/-) mice. CB2 receptor KO mice were created by
breeding heterozygote CB2 (+/-) mice (Buckley et al., 2000).

Double KO (CB1 (-/-)/CBZ (-/-) mice) were obtained by breeding CB1 (-/-) mice
with CB2 (-/-) mice to create double heterozygote CB1 (+/-)/CB2 (+/-) mice. Double
heterozygote mice were then bred with one another to obtain CB1 (-/-)/CB2 (-/-) mice
which were continually bred with one another to maintain a double KO mouse colony
(Jarai et al., 1999). CB 1 (-/-)/CB2 (-/-) were originally obtained by Drs. Kaminski and
Kaplan from Dr. Andreas Zimmer of the University of Bonn, and were used to establish a
breeding colony at Michigan State University.

CB1 (-/-)/CB2 (-/-) mice were genotyped using tail samples. Approximately 1 cm
of the terminal tail was digested using 0.5 mg of Proteinase K (Roche, Basel,
Switzerland) in nuclei lysis buffer (25 mM ethylenediarninetetraacetic acid (EDTA), 75
mM NaCl) incubated overnight in a water bath at 58°C with gentle shaking. The
following day digested samples were vortexed to homogenize remaining tissue and 30 ug
of RNase (Roche) was added to tubes containing lysates and inverted 5 times. Lysates
were incubated for 30 min at 37°C in a water bath. Next, 200 111 of protein precipitation
solution (4.21 M NaCl, 0.63 M KCl, 8 mM Tris-HCl, pH 8.0) was added to 650 uL of

lysate, vortexed for 20 s, chilled on ice for 5 min, and centrifuged for 4 min at 16,000 x g

54

at 4°C in a Microfuge 18 Centrifuge (Beckrnan Coutler Inc., Fullerton, CA).
Supematants were removed and placed in new tubes with 600 uL of isopropranol and
inverted until DNA strands could be visualized. Samples were centrifuged at room
temperature for 1 min to pellet DNA, which was resuspended in 600 uL of ethanol and
washed by inversion. Samples were re-centrifuged at room temperature to pellet DNA,
followed by an additional ethanol wash. Ethanol was decanted from tubes, DNA pellets
dried, and incubated in 100 uL of DNA rehydration solution (1 mM EDTA, 10 mM Tris-
HCl) for 1 h at 65°C in a water bath. DNA concentration was determined using a
NanoDrop 1000 Spectrophotometer (Therrno Scientific, Wilmington, DE) and further
diluted in DNA rehydration solution to a final concentration of 50 ng/uL.

Polymerase chain reaction (PCR) was used to confirm knockout of CB] and
CBZ receptor genes. PCR exponentially amplified base pair products of the CB1 or CB2
genes using 3’ and 5’ primers (Table 2-1) specifically targeted to the mouse CB1 and
CB2 receptor genes in the presence of a DNA polymerase and deoxynucleotide
triphosphates (dNTP’s). As a positive control, primers targeting the neomycin cassette
inserted into the CB2 receptor gene were used to amplify a product of the neomycin
cassette. DNA expression of CB1 and CB2 receptor transcripts in WT mice was
determined using 250 ng of genomic DNA isolated from tail samples in a volume of 5
uL. A master mix of PCR reagents (1x PCR reaction buffer, 1 mM MgC12, 25 U/uL Taq
DNA Polymerase, 0.2 mM dNTP’s, and 0.2 mM of each forward and reverse primers for
either CB1 WT positive or CB2 WT positive and CBZ KO positive) was made and 20 uL

of master mix was added to the 5 uL of genomic DNA (50 ng/uL).

55

Table 2-1. PCR Primers for GB] and CB2 Receptors and CB2 KO Neomycin
Cassette

 

Primer Sequence

 

 

 

 

 

 

CB1 (+/+)

Forward 5'-AGGAGCAAGGACCTGAGACA-3'

Reverse 5'-GGTCACCTTGGCGATCTTAA-3'

CBZ (+/+)

Forward 5'-CCTGATAGGCTGGAAGAAGTATCTAC-3'
Reverse 5’-ACATCAGCCTCTGTTTCTGTAACC-3'
CB2 (-/-)

Forward 5'-ACCGCTGTTGACCGCTACCTATGTCT-3’
Reverse 5'-TAAAGCGCATGCTCCAGACTGCCTT-3'

 

Table 2-1. PCR primers for CB1 and CB2 receptors and CB2 KO neomycin cassette. If
a gene of interest was present, primers produced a 183 base pair (bp) product of the CB1
gene, a 93 bp product of the CB2 gene, or a 359 bp product of the CB2 KO neomycin
gene cassette.

56

PCR reaction was initiated in a PX2 Thermo Hybaid PCR machine (Thermo
Scientific) using 40 cycles of 45 s at 94°C for denaturation of DNA, 30 s at 55°C for
annealing, and 90 s at 72°C for extension. Amplified cDNA was then stored at -20°C
until 10 uL of amplified DNA was run on a 2% agarose gel (75 V for 1 hr) to determine
DNA transcript knockout of CB1 and CB2 receptor genes in C81 (-/-)/CB2 (-/-).
Amplified DNA was visualized on gels using a UV transilluminator (UV C Co., San
Gabriel, CA) to confirm knockout of CB1 and CB2 receptor genomic DNA in knockout

mice and presence in WT C57BL/6 mice (Figures 2-1 and 2-2).

57

 

WI'KO

Figure 2-1. CBl receptor product in WT mice, but not CB1/CB2 KO mice. WT and
CBl/CB2 KO mice were genotyped to confirm absence of the CB1 receptor as revealed
by lack of a CB1 receptor PCR product at 183 bp in CBl/CBZ KO mice seen in WT
mice.

58

Neomycin
Cassette 359 bp —-—> ’1

cm 93 bp ———>

 

Figure 2-2. CB2 receptor product in WT mice, but not CBl/CB2 KO mice. WT and
CB1/CB2 KO mice were genotyped to confirm KO of the CBZ receptor as revealed by
lack of a CB2 receptor PCR product at 93 bp seen in WT mice. Mice were also
genotyped for the neomycin cassette inserted into the CBZ receptor gene of CB2 KO
mice, which produced a 359 bp PCR product observed only in CB1/C82 KO mice.

59

Animal Housing and Maintenance

All animals were provided with food and water ad libitum, and housed
individually or in groups of up to 5 mice per cage at 25°C with a 12 h light/dark cycle
(lights on: 06:00). Mice receiving MPTP were housed in a separate room from saline
treated control mice to prevent any contamination of saline treated mice with bedding or
waste of MPTP treated mice. Mice that underwent stereotaxic surgery receiving vehicle
or LPS were housed individually but within the same room.

All experiments used the minimal number of animals required, minimized
suffering, and followed the guidelines of the National Institutes of Health Guide for the
Care and use of Laboratory Animals. All drug treatments, surgical procedures, and
methods of euthanasia were approved by the Michigan State Institutional Animal Care
and Use Committee (IACUC). Animal use form (AUF) numbers for drug treatment and
surgical procedures are: Sildenafil, apocynin, and MPTP treatment experiments AUF #:
08/04-112-00. CBl/CB2 K0 and stereotaxic LPS surgery and peripheral LPS treatment

experiments AUF#: 05/07-076-00.

60

B. Drugs
Saline or Vehicle Treatment
All saline or vehicle treatments were given as a 10 mL/kg volume either given

subcutaneously (so) or intraperitoneally (i.p.).

MPT P

MPTP-hydrochloride (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.9%
NaCl (saline). MPTP was administered in one of four different treatment paradigms with
0.9% saline serving as the vehicle control in all cases. All MPTP dosing was expressed
as mg of MPTP free base per weight of the animal. One of 4 different MPTP dosing
paradigms was used for experiments; acute, sub-chronic, chronic/probenecid, or repeated
acute MPTP treatment (Table 2-2). MPTP was injected so for acute, sub-chronic, and
chronic/probenecid experiments, and i.p. for repeated acute treatment. For
chronic/probenecid MPTP experiments probenecid (Sigma-Aldrich) was administered via

i.p. injection.

61

Table 2-2. MPTP Treatment Paradigms

 

 

MPTP Paradigm MPTP Frequency
Dosage
Acute MPTP 10 mg/kg Once
Sub-Chronic MPTP 10 mg/kg Once a day for 5 days
Chronic MPTP 20 mg/kg One injection of MPTP and one injection of
with Probenecid probenecid (250 mg/kg) every 3.5 days for

5 weeks for a total of 10 injections,
probenecid injection was given 1 h prior to
MPTP administration

Repeated Acute 16 mg/kg One injection of MPTP every 2 h over a 6 h
period for a total of 4 injections and

cumulative MPTP dosage of (64 mgkg)

62

Sildenafil

Sildenafil citrate (Pfizer, Ann Arbor, MI) was dissolved in 0.9% saline to a
concentration of either 0.5 or 1 mg/mL. Mice received sildenafil 5 or 10 mg/kg so. every
12 h beginning 3 days prior to MPTP treatment (Pre-treatment), 3 days following the
initiation of MPTP treatment (Concurrent treatment), or 3 days following the final MPTP
injection (Post-treatment). Sildenafil administration continued daily from the point of
initiation to the morning of sacrifice with the last injection given 30 min prior to

decapitation.

Apocynin Administration

Apocynin (Sigma-Aldrich) was prepared as a 6 mg/mL solution dissolved in 0.1
N sodium hydroxide (NaOH), brought to a pH of 8.0 using 1 M Tris-HCl (pH 6.8) and
q.s. to an appropriate volume with ddeO. Vehicle solution used as a control for
apocynin was prepared as described for apocynin minus the drug. For the sub-chronic
MPTP experiment mice received an injection of 100 mg/kg apocynin i.p. or vehicle 2 h
prior to injection of 0.9% saline or MPTP, and a second injection of apocynin or vehicle
12 h later. Apocynin administration was initiated 2 days prior to the first MPTP injection
and continued twice daily up to and including the day of sacrifice, which was 3 days
following the last MPTP injection.

For the repeated acute MPTP experiment apocynin was constantly infused via an
Alzet osmotic mini-pump 1007D (Durect, Cupertiono, CA) beginning 3 days prior to
initiation of MPTP treatment. Apocynin was dissolved in 50 % dimethyl sulfoxide

(DMSO) and 50% polyethylene glycol (PEG) to a concentration of 48 mg/mL apocynin.

63

The osmotic mini-pump delivered 0.26 uL/h of a vehicle 50% DMSO/50% PEG or 48
mg/mL apocynin solution. This infusion rate allowed the delivery of 250 mg/kg
apocynin per day for a mouse weighing 30 g. Pumps were filled with either vehicle or
apocynin solution, and placed in 0.9% saline for 18 h to prime pumps before so
implantation.

Mice were anesthetized with 80 mg/kg Ketamine/ 12 mg/kg Xylazine for sedation
for pump implantation. Following sedation the incision site was cleaned with betadine
before a midline so one inch incision was made between the scapulas. Pumps were then
placed underneath the skin, and the surgical site was closed with surgical staples. Mice
normally recovered from sedation within 1-2 h following Ketamine/Xylazine

administration.

Raclopride
The D2 receptor antagonist raclopride (Si grna-Aldrich) was dissolved in 0.9%
saline at a concentration of 0.1 mg/mL. Raclopride (1 mg/kg) or 0.9% saline vehicle was

injected i.p. 1 h prior to sacrifice.

y-Butyrolactone (GBL) and Quinelorane

GBL (Sigma-Aldrich) which inhibits firing of DA neurons was diluted to a 0.75
mg/mL solution in 0.9% saline from a 1 g/ml stock solution and quinelorane (Sigrna-
Aldrich) was dissolved in 0.9% saline to concentration of 0.01 mg/mL. Saline vehicle or

the D2 receptor agonist quinelorane 0.1 mg/kg was given to mice i.p. 1 min prior to either

64

saline or GBL 750 mg/kg i.p. administration and mice were sacrificed 1 h following

saline or quinelorane administration.

CB Receptor Antagonists

The CB1 receptor antagonist, rimonabant (SR141716A), and the CB2 receptor
antagonist, SR144528 both obtained from the National Institute of Drug Abuse (NIDA)
by Dr. Norbert Kaminski were individually dissolved in a solution of 10% Tween-80,
10% Ethanol, and 80% saline. The 10% Tween-80/ 10% Ethanol/ 80% saline served as
the vehicle solution for both rimonabant and SR144528. Mice were injected once daily
so. 1 h prior to 0.9% saline or MPTP administration with either the vehicle solution,
rimonabant 1 mg/kg, SR144528 2.5 mg/kg, or both rimonabant 1 mg/kg and SR144528
2.5 mg/kg. Mice continued to receive either the appropriate vehicle solution or one or
both CB antagonists once daily in the morning each day following the termination of
0.9% saline or MPTP administration until 3 days following the last MPTP injection when

mice were decapitated.

Lipopolysaccharide (LPS)

LPS or bacterial endotoxin can induce significant activation of microglial cells
within the brain inducing localized or widespread inflammation within the brain
depending on the method of delivery. In order to induce localized inflammation within
the substantia nigra, LPS was injected unilaterally into the left side of the substantia nigra
as described below. Alternatively, inflammation throughout the brain was initiated with

the injection of LPS intraperitoneally.

65

Unilateral Stereotaxic LPS Injection into the Mouse Substantia Nigra

LPS or phosphate buffered saline (PBS) (0.14 M NaCl, 3.4 mM NaHzPO4, 6.8
mM NazHPO4, pH 7.4) vehicle was injected unilaterally into the left substantia nigra to
induce localized inflammation. Mice were lightly sedated with 50 mg/kg Ketamine/2.5
mg/kg Xylazine and approximately 15 min later were completely anesthetized with
isoflurane using a Vapomatic Vaporizer (A. M. Bickford, Wales Center, NY). The
surgical area was shaved and the mouse was positioned in a stereotaxic apparatus under
continuous isoflurane flow via a modified nose cone mask. The surgical area was
cleaned with alcohol and betadine, a midline incision was made in the scalp along the
mid-saggital line, the skull was exposed, and both Bregrna and lambda were marked on
the surface of the skull with a pencil.

Vertical measurements were taken at bregrna and lamda using a 30 gauge blunt
tipped needle attached to the stereotaxic apparatus. If vertical measurements were not
equivalent at both of these points the toothbar was adjusted appropriately to make the
measurements equivalent. Medial/lateral and anterior/posterior plane measurements were
taken in reference to Bregma to determine the coordinates for stereotaxic injection. The
injection needle was raised from the surface of the skull and positioned to +1.3 mm
(lateral/medial plane) and -3.0 mm (anterior/posterior plane) with regard to Bregrna,
coordinates corresponding to location of the left substantia nigra (Franklin and Paxinos,
1996). A dremel drill was used to drill a hole through the skull at the site of injection.
The blunt tipped 30 gauge needle was flushed with PBS and either PBS (vehicle) or LPS
was drawn up through the needle/tubing attached to a 1 uL Hamilton syringe. The needle

was lowered into the skull -4.7 mm (dorsal/ventral plane) with regard to Bregrna.

66

The specified volume of PBS or LPS 2.5, 5, or 10 ug (Sigma Aldrich) was
injected into the substantia nigra over a period of 2 min. LPS was obtained from
escherichia coli 01 11:B4 (catalog #: L3012-10 mg batch #: 067K4139, L3012-5 mg
batch #: 047K4089, and L2880-100 mg batch #: 066K4039, Sigma-Aldrich). The needle
was left in place for 5 min following the period of PBS/LPS injection before being
removed from the brain to reduce backflow of PBS/LPS. The hole in the skull was
closed using bone wax and the scalp sutured using wound clips. Mice were carefully
observed for any signs of pain or distress over the entire time period following surgery
until they were euthanized. Mice did not receive any anti-inflammatory agents as this

would interfere with the inflammatory effects of LPS in the brain.

Peripheral LPS Administration

Mice received either 0.9% NaCl vehicle or LPS 5 mg/kg (Sigma Aldrich)
administered in a 0.5 mg/mL LPS solution via i.p. injection 1 h prior to sacrifice by
decapitation. LPS was obtained from escherichia coli 01 1 1 :B4 (catalog # L3012-5 mg

batch #: 047K4089, Sigma-Aldrich).

67

C. Brain Tissue Preparation
Euthanasia

Mice were killed by one of two methods for all experiments. Mice were
decapitated using a guillotine for collection of brain tissue used for either neurochemistry
and/or Western Blot analysis. For immunohistochemistry analyses mice were given a
lethal dose of Ketarnine 266 mg/kg and Xylazine 4 mg/kg and perfused with saline
followed by 4% paraformaldehyde. Brains were subsequently removed and placed in 4%

paraformaldehyde for at least 24 h, then stored in 20% sucrose for cryoprotection.

Preparation of Brain Tissue

After mice were sacrificed by decapitation for neurochemistry and Western blot
analyses the brain was immediately removed and the median eminence was dissected
from the brain and placed in 50 uL of tissue buffer (0.05 M sodium phosphate, 0.03 M
citrate, 15% methanol, pH 2.5). The median eminence was stored at —20°C until
preparation for neurochemical analysis. Whole brains were immediately frozen on dry
ice and stored at -80 °C until they were sectioned in a cryostat at —10°C (HM525
Microtome Cryostat, Microm International or CTD-Model Harris, International
Equipment Co., Needham, MA).

Brain tissue used for immunohistochemistry analysis was fixed in 4%
paraformaldehyde either by drop fixation or perfusion. For drop fixation mice were
decapitated, the brain removed, and dissected at the mid-coronal plane through the
hypothalamus. The caudal portion of the brain (hindbrain) was placed in 4%

paraformaldehyde for 7 days and stored at 4°C. Hindbrains were subsequently

68

cryoprotected in 20% sucrose following paraformaldehyde incubation. Mice perfused
with 4% paraformaldehyde were decapitated following perfusion, brains removed and
placed in 4% paraformaldehyde and stored at 4°C for 24 h. Brains were later placed in
20% sucrose for at least 24 h before sectioning. Both drop fixed and perfused brains

were sectioned in a cryostat at -25°C.

69

D. Assay of Amines, Amine Metabolites, and Apocynin in Brain Tissue

Amines and apocynin were measured in brain extracts using high performance
liquid chromatography with amperometric electrochemical detection (HPLC—EC).
HPLC-EC measures amines and apocynin by oxidation at an electrode held at a constant
voltage or potential. The oxidation of an amine or apocynin at the electrode produces a
current that is proportional to the concentration of compound in the brain extract. In
order to detect an amine or apocynin the potential must be high enough to oxidize the
compound. The optimal potential for oxidation of amines is 0.4 V (Eaton et al., 1994),
but no information is available for apocynin. Accordingly, the ability of apocynin to be
oxidized at various potentials was examined and the resulting voltamogram is depicted in
Figure 2-3 (see section below entitled Measurement of Apocynin within the Brain). The
optimal voltage to measure apocynin was determined to be 0.47 V.

The higher the current produced as the brain extract passes through the electrode
detector, the more of the compound of interest is present in the brain extract, as reflected
by a higher peak height. By comparing peak height of a standard to the peak height of a
compound in a brain extract, the concentration of the compound within a sample can be
calculated. Different compounds that are oxidized at similar potentials can be
distinguished by the retention time of compound on a C-18 column. The more
hydrophobic a compound is the longer it will remain on the column and the longer the

retention time of the compound.

70

500
450
400
350
300
250
200
150
100

50

Peak Height x 10,000

 

A A - - A -

+.40 +.41 +.42 +.43 +.44 +.45 +.46 +.47 +.48 +.49 +.50
Vouage

Figure 2-3. Apocynin voltamogram. Apocynin (25 ng) standards were measured at a
sensitivity of 10 x 50 and voltages ranging from +0.40 to +0.50 using HPLC-EC to
determine an ideal voltage to detect apocynin.

71

Tissue Collection and Preparation

Frozen brain tissue was sectioned coronally in a cryostat at -10 °C at 500 uM
going from the rostral to caudal brain beginning at approximately Bregma +1.94 mm
(Franklin and Paxinos, 1996). Brain tissue sections were mounted directly onto slides
and slides were placed on dry ice. Brain regions including the striatum and nucleus
accumbens were microdissected under a Stereo Master Microscope (Fisher Scientific,
Pittsburgh, PA) at 4x magnification using a modification of the Palkovits method
(Palkovits and Brownstein, 1983). The nucleus accumbens was sampled using a 21
gauge (0.5 mm inner diameter) circular punch tool and striatum an 18 gauge (1 mm inner
diameter) circular punch tool. Tissue punch sites for specified brain regions are pictured
in (Figure 2-4). Tissue punches were placed in 50 uL of tissue buffer and stored at -20°C
until preparation for neurochemical analysis.

Samples were thawed and centrifuged for 30 s at 18,000 x g in a Microfiige 18
Centrifuge (Beckrnan Coutler Inc., Fullerton, CA) and tissue was sonicated with three 1 s
bursts (Heat Systems Ultrasonics, Plainview, NY). Tissue samples were again
centrifuged for 30 s at 18,000 x g to pellet tissue. Tissue buffer containing
neurochemicals released with sonication was collected using a beveled tip 100 uL
Hamilton syringe and q.s. using tissue buffer to a final volume of 100 uL for striatum
samples and 65 uL for median eminence and nucleus accumbens samples. Samples were
placed in new tubes for analysis of neurochemicals using high performance liquid
chromatography with electrochemical detection (HPLC-EC) (Eaton et al., 1994).

‘ Remaining tissue pellets were placed in 100 uL of l N sodium hydroxide

(N aOH). Tissue pellets were sonicated and vortexed to homogenize samples. Protein

72

Striatum

 

 

Nucleus Accumbens

Figure 2-4. Diagram showing striatum and nucleus accumbens sampling sites. Bilateral
samples were taken from both the striatum and nucleus accumbens using an 18 gauge (1
mm inner diameter) and 21 gauge (0.5 mm inner diameter) circular punch tool
respectively. Pictorial coronal section of the mouse brain at Bregrna +1.42 (Franklin and
Paxinos, 1996).

73

content of tissue samples was determined using a Lowry protein assay (Lowry et al.,
1951). Samples were reacted with 1 mL of reagent A solution (0.18 M sodium carbonate,
0.63 mM cupric sulfate, 0.95 mM KNA tartrate) for 10 min, and with 100 uL of reagent
B solution (2 N Folin-Ciocalteau’s phenol reagent diluted 1:1 with ddH20) for 30 min to
produce a colorimetric reaction. Protein content was determined using a standard curve
of 0, 12.5, 25, and 50 ug bovine serum albumin (BSA) protein (Sigma) dissolved in 1 N
NaOH. The absorbance of standards and samples was read at 700 nm using a U-Quant
plate reader (BioTek Instruments Inc., Winooski, VT). Protein content in samples was

quantified by normalizing sample absorbance to absorbencies of protein standards.

Measurement of Amines and Amine Metabolites in the Brain

Content of DA, DOPAC, HVA, 5HT, and 5HIAA in samples was determined
using HPLC-EC. A Waters 515 HPLC pump was used to circulate the HPLC-EC mobile
phase (0.05 M sodium phosphate, 0.03 M citrate, 0.1 mM EDTA, pH 2.65) at a flow rate
of 1 mL/min through an ESA Coulochem 5100A electrochemical detector set at +0.4
volts. Brain sample extracts were injected onto a C18 reverse phase analytical column
(Bioanalytical Systems, West Lafayette, IN). Standards (1 ng) of DA, DOPAC, HVA,
5HT, and SHIAA were used to determine brain tissue neurochemical content. Separation
of compounds was achieved using 15-25% methanol and 0.02-0.05% sodium
octylsulfate. Peak heights of standards (Figure 2-5; Panel A) were used to determine
neurochemical content in brain samples (Figure 2-6; Panel‘s A & B) using sample peak
heights. Neurochemical sample content was normalized to protein content of samples

and expressed as ng of neurochemical per mg of protein.

74

Measurement of Apocynin within the Brain

Tissue punches of the striatum were taken from 500 um brain tissue sections
using an 18 gauge punch tool and placed in tissue buffer. Striatum tissue punches were
sonicated with three 1 s bursts, centrifirged for 30 s at 18,000 x g to pellet tissue, and
supematants were removed and q.s. using tissue buffer to a final volume of 100 uL.
Apocynin was measured using HPLC-EC by injecting striatum supernatant extracts onto
a C18 reverse phase analytical column coupled with BSA Coulochem 5100A
electrochemical detector set at +0.47 V. The optimal voltage to measure apocynin was
determined by measuring 25 ng apocynin standards at specified voltages between (+0.4 V
to +0.5 V) creating a voltarnaograrn (Figure 2-3). Apocynin concentration was minimally
detected at lower voltages but the standard curve of the voltamogram was relatively
linear to about +0.47 V which there after the curve became exponential (Lookingland,
Goudreau and Sayed, unpublished results). Apocynin content in samples was quantified
by comparing sample peak heights (Figure 2-6; Panel C) to standards (Figure 2-5; Panel
B) and normalized to tissue sample protein content determined using a Lowry protein

assay (Lowry et al., 1951).

Measurement of MAO B Activity in vitro

MAO B activity was measured in vitro to determine if apocynin inhibits MAO B
metabolism of DA to DOPAL. The synthesis of DOPAL from DA in the presence of
MAO B (Sigma-Aldrich) was measured at 15, 30, 45, and 60 min following the initiation
of the reaction. The reaction solution contained 5 uM DA in phosphate buffer (pH 7.5)

and synthesis of DOPAL was initiated by the addition of 2.8 units of MAO B to 250 uL

75

of the reaction solution. MAO B enzyme activity was stopped at the indicated time
points with the addition of tissue buffer (100 uL) for every 50 uL of the reaction mixture.
The concentrations of DA and DOPAL were measured in reacted samples using HPLC-
EC (Figure 2-6, Panel D) as described in this section (Measurement of Amines and
Amine Metabolites in the Brain).

The synthesis of DOPAL was also measured in the presence of apocynin 60 min
following the addition of MAO B to the reaction mixture to determine if apocynin
inhibited MAO B activity by decreasing the synthesis of DOPAL. The reaction mixture
contained 5 uM DA in phosphate buffer (pH 7.5) and apocynin (1, 10, or 100 uM), and
synthesis of DOPAL was initiated by the addition of 2.8 units of MAO B to 250 uL of the
reaction mixture. The reaction was terminated 60 min later as described above and DA

and DOPAL concentrations determined using HPLC-EC.

76

 

 

 

 

 

 

A.
A DOPAC
2- DA HVA
a l 5HIAA/
C
V 5HT
E /
.9 1
O
I
.2
N
0 I
D.
111 titer
.1

 

 

10 15 20 25

Peak Height (namp)

Time (min)

 

 

 

 

Apocynin

10 15 20 25

Figure 2-5. Chromatograms show peak height and retention time for the respective
neurochemicals or apocynin. Representative HPLC-EC Chromatograms for 1 ng DA,
DOPAC, HVA, SHIAA, and 5HT standards (Panel A) and 25 ng apocynin standard

(Panel B).

77

[j
10 1s 20 25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

< l
_. °\ :3} __
Er” ”3r:
D (dweunufiiaH need
5% —8
o/ o "8
2 , g :4 ~—.«2
\ V \fi “35 —2
- a: .__, 3.?
e be é
, (dweulrufiiaH need 0
0 .. .'s'
_N '_
or _.32
8\ 3
1 ::
(dweu) lll5!9H need
“5 —a
g E ”if; ‘8
< (dweunlifiiaH need

Figure 2-6. Chromatograms showing peak height and retention times for DA, DOPAC
and apocynin in striatum samples. Representative HPLC chromatograms of DA and
DOPAC in the striatum obtained from saline (Panel A), sub-chronic IVIPTP treated (10
mg/kg once a day for 5 days; Panel B), and apocynin (100 mg/kg 2.5 min prior to
decapitation; Panel C) mice. Representative HPLC chromatogram of DA and DOPAL 30
min following initiation of DA metabolism by MAO B (Panel D).

E. cGMP Assay

cGMP concentrations were measured in the brain to determine if sildenafil
inhibition of PDES increased cGMP levels in the brain. Following decapitation the brain
was removed and sectioned mid-coronally. The hindbrain was frozen on dry ice,
weighed, and placed in liquid nitrogen. Brain tissue was homogenized in liquid nitrogen
with a mortar and pestel, and placed in a 1.5 mL tube with 0.1 N HCl with 1 mM of 3-
isobutyl-l-methylxanthine (IBMX) in a 20% weight per volume solution and vortexed to
disperse tissue within solution. Samples were centrifirged for 15 min at 600 x g at 4°C
and supematants were removed and assayed for cGMP content. Pelleted brain tissue was
resuspended in 800 uL of 1 N NaOH and protein content determined using a Lowry
protein assay (Lowry et al., 1951). cGMP content of supematants were determined in
duplicate as per directions of the acetylation method of the low cGMP kit (R&D Systems,
Minneapolis, MN). cGMP content of individual samples was determined by
extrapolation from a standard cGMP curve (Figure 2-7). Colorimetric reaction of
individual samples used to determine cGMP content was read on U-Quant plate reader at

405 nm with background subtraction of 570 nm.

79

Brain cGMP Standard Curve

 

 

3 0 20 . y = -0.0251Ln(x) + 0.0909
6 ' R2 = 0.936
;' 0.15 i
0
g 0.10 -
‘2 0.05.
3
< 0.00 I l 1
0.01 0.10 1.00 10.00 100.00
cGMP (pmollmL)

Figure 2-7. cGMP standard curve. Standard cGMP concentrations were plotted against
the mean of duplicate standard absorbances (optical density or CD.) The cGMP
concentration of samples was extrapolated by the use of the linear equation of the line
obtained by known cGMP concentration of standards to standard absorbances.

80

F. Plasma Sildenafil Determination

Trunk blood was collected following decapitation and placed in heparinzed tubes
for analysis of free Sildenafil levels. Plasma was isolated and collected from blood by
centrifugation of heparinzed tubes at 2,000 rpm for 20 min at 4°C in a 3000R Centrifuge
(F isher-Scientific). The concentrations of free Sildenafil in plasma were determined 30
min after a single (5 or 10 mg/kg s.c.) Sildenafil dose. Concentrations of free Sildenafil in
plasma were measured by Pfizer (Sandwich, United Kingdom) using HPLC with
ultraviolet detection (UV). UV detection of Sildenafil was obtained at an absorbance of

290 nm (Walker et al., 1999).

81

G. Western Blot Analysis

Western blotting was used to determine the TH content of NSDA neuronal cell
bodies and proteins (TH, serine 40 phosphorylated TH, and DAT) in axon terminals. TH
and DAT protein content were used as indices of NSDA neuronal cell body and or
terminal integrity. Proteins associated with microglial cells (p67Phox, gp91Phox) and
dopaminergic neurons (DA and CAMP regulated phosphoprotein; DARPP-32) and alpha-
synuclein were also measured by Western blot. All proteins of interest were normalized
to homogenously expressed proteins (B-III tubulin, synaptosomal-associated protein 25
(SNAP-25), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) to control for

protein loading variations.

Brain Tissue Preparation

Brains were sectioned at 500 um in a cryostat at -10°C and mounted onto room
temperature slides. Striatum tissue was obtained by taking two 12 gauge tissue punches,
or ventral midbrain tissue was dissected from coronal brain sections with a razor blade
(Figure 2-8). Brain tissue was placed in a sucrose homogenization (0.25 M sucrose, 10
mM HEPES, 10 mM MgC12, pH 7.4) or a lysis buffer (1% sodium dodecyl sulfate (SDS),
20 mM Tris-HCl, pH 7.4). Cytosolic and membrane protein fractions were obtained
using one of two serial centrifugation methods.

One method of isolating membrane and cytosolic fractions of brain tissue used
was a serial centrifugation and sucrose gradient method (Figure 2-9) (Leng et al., 2001).
Tissue was sonicated and centrifuged for 10 min at 500 x g at 4°C to pellet membrane,

nuclear, and cellular debris (P1). The supernatant (51) was removed and centrifuged

82

Striatum

 

Ventral Midbrain

Figure 2-8. Pictorial diagram of striatum and ventral rrridbrain tissue sites taken by tissue
punch or dissection. Striatum tissue punch sites (Panel A) and ventral midbrain area
tissue (Panel B) dissected for Western blot, NADPH oxidase assay, and or Complex 1
activity assay. Striatum tissue punches were taken using a 12 gauge punch tool (1 mm
inner diameter) and ventral midbrain tissue was dissected with a razor blade. Pictorial
coronal sections of the mouse brain at Bregma +1.18 (striatum) and -3.08 (ventral
midbrain) (Franklin and Paxinos, 1996).

83

at 18,000 x g for 20 min at 4°C. The supernatant (S2) obtained from the second
centrifugation step was used to analyze cytosolic protein content. The pellet (P1) from
the first centrifugation was resuspended in 200 uL of sucrose homogenization buffer. A
membrane fraction was isolated by layering resuspended (Pl) samples on top of 330 uL
of a high density sucrose buffer (2.4 M sucrose, 10 mM HEPES, 10 mM MgC12, pH 7.4),
and centrifirging samples for 7.5 h at 16,000 x g at 4°C. Following centrifugation the
membrane fraction layer was located between the low and high density sucrose buffers
and was drawn up in a volume of about 150 uL. Protein content of cytosolic and
membrane fractions was determined using a bicinchoninic acid (BCA) protein assay.
The BCA assay determines sample protein content by reacting 5 uL of tissue homogenate
and 95 uL of ddeO with 2 mL of a protein reaction solution (50:1 bicinchoninic acid:
copper (Cu) 11 sulfate). The BCA assay uses protein reduction of Cu 11 to Cu I in a
protein concentration dependent manner, followed by the reaction of Cu I with
bicinchoninic acid, a Chromogen which was measured at 562 nm on a Beckman DU640
spectrophotometer (Beckman Coulter). Protein sample concentration was quantified
using absorbencies of BSA protein standards of 2.5, 5, 10, 15, and 20 ug/uL.

Cytosolic and membrane fractions of brain tissue were also isolated using an
ultracentrifugation method (Guillemin et al., 2005; Amadesi et al., 2006). Although the
sucrose gradient method of membrane isolation was successful, the protein concentration
per uL of membrane homogenate obtained using this method yielded low protein content.
This problem was addressed by developing an ultracentrifugation method of cytosolic
and membrane fraction isolation (Figure 2-10). Striatum and ventral midbrain tissue was

taken, placed in 300 uL of lysis buffer, and sonicated to form a homogenous sample.

84

Sucrose Gradient Protein Fractionation

 

 

 

 

U

Homogenized Brain Tissue

1....

 

S1

 

 

Rosuspondod P1

 

 

Sucrose Gradient P1
16.000 x /
Membrane

Fraction _ U

 

 

18,000 x g

 

Cytosolic

$2 "_" Fraction

 

 

 

P2

Figure 2-9. Pictorial diagram of sucrose gradient fi'actionation of membrane and
cytosolic fractions. Homogenized brain tissue was centrifuged at 500 x g for 10 min.
The supernatant (S1) was centrifuged at 18,000 x g for 20 min to obtain the S2 cytosolic
fraction, and pellet (P1) was resuspended in sucrose homogenization buffer. The P1
pellet was layered onto a higher density sucrose buffer and centrifuged at 16,000 x g for
7.5 h to obtain a membrane fraction at the interface of the lower and higher density

sucrose buffers.

85

Ultracentrifugation Protein Fractionation

 

Homogenized Braln Tissue

 

 

 

16,300 x g

 

61
P1

1

S1

 

 

 

 

 

 

 

1 130,000 RPM

 

Cytosolic
Fraction

$2

 

 

 

Membrane
Fraction —"’ P

Figure 2-10. Pictorial diagram of isolation of membrane and cytosolic fractions using
ultracentrifugation. Brain tissue was centrifuged at 6,300 x g for 10 min to obtain the
supernatant (SI) and pellet (P1) fractions. S1 was centrifuged at 130,000 RPM to obtain
the S2 (cytosolic) and P2 (membrane) fractions.

86

Samples were then centrifuged for 10 min at 6,300 x g at 4°C to pellet cellular and
nuclear debris (P1). The supernatant (S1) fiom this initial centrifugation was removed
and centrifuged using a T-120 rotor in an Optima Max Ultracentrifuge (Beckrnan
Coutler) at 130,000 rpm for 30 min at 4°C. The supernatant (S2) fiom this centrifugation
step was used as the cytosolic fraction and the pellet (PZ) contained the membrane
fraction and was resuspended in 50 uL of PBS. Protein content for all samples was
determined using a BCA protein assay. Protein samples were prepared for
electrophoresis by the addition of 1:10 or 1:4 dilution of loading dye (0.25 M Tris-HCl
pH 6.8, 5% SDS, 0.05% Bromophenol blue, 45% glycerol, and 2-mercaptoethanol).

Protein samples were boiled at 95-100°C for 5 min to denature proteins.

Electrophoresis and Transfer of Proteins

A total of 20-80 ug of protein/well was loaded on one of the following types of
precast gels: 7.5%, 10%, 4-15%, or 4-20% Tris-HCl polyacrylamide gels (Bio-Rad
Laboratories, Hercules, CA) with 10-15 wells for electrophoresis. The percentage of
polyacrylamide in gels was adjusted depending on the molecular weight of the proteins of
interest. A protein standard ladder was loaded to determine protein molecular weight of
specific proteins of interest. The inner chamber formed by two precast gels placed within
the mini-protean 3 cell (Bio-rad) was filled with electrophoresis running buffer (50 mM
Tris-base, 0.38 M glycine, 3.5 mM SDS), and another 300 mL was placed outside of the
Chamber. Protein samples were separated by running 150 V through the mini-protean 3
cell until the lowest molecular weight protein standard reached the bottom of the each

gel. Gels were removed from the electrophoresis chamber and placed on a 0.45 pm

87

nitrocellulose membrane for transfer of proteins using a trans-blot cell (Bio-rad). The
trans-blot cell was filled with transfer buffer (25 mM Tris-base, 0.19 M glycine, 20%
methanol) and 300 mV was applied for 90 min to transfer proteins to the membrane.
Membranes were washed in Tris buffered saline (TBS) (50 mM Tris-base, 2.7 mM KCl,
0.14 M NaCl, pH 7.4) 3 x 5 min, and non-specific proteins on membranes were blocked
using Oddyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE) for 1 h.
Membranes were probed overnight for specific proteins including TH, phosphorylated
serine 40 TH, p67Phox, gp91Phox, DAT, a-synuclein, DARPP-32, parkin, SNAP-25,
GAPDH, and B-III tubulin using primary antibodies developed in rabbit, mouse or rat
(Table 2-3). Primary antibodies were reacted with an appropriate secondary antibody

with an infrared fluorescent tag for 1 h (Table 2-3).

Quantification of Protein Content and Normalization to Control Proteins

Proteins tagged with primary and secondary antibodies were visualized using an
Oddyssey infrared imaging system (LI-COR Biosciences) which uses infrared
fluorescence detection of secondary antibodies at either 700 or 800 nm to create a digital
image of protein bands. Protein content was determined using densitometry analysis of
digital images Protein content of TH, phosphorylated serine 40 TH, p67Phox, gp91Phox,
DAT, a-synuclein, parkin, or DARPP-32 was normalized to cytosolic (GAPDH or B-III
tubulin) or the membrane protein SNAP-25 all three of which are proteins homogenously
expressed in brain tissue. Individual proteins normalized to cytosolic or membrane

proteins are expressed as relative density units (RDU).

88

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Protein Species Dilution Source
Tyrosine Rabbit 1 :2,000 Millipore.
hydroxylase (TH) Billerica, MA
iAB152)
Phosphoryiated Rabbit 1 :1 .000 Cell Signaling
Serine 40 TH Danvers, MA
(2791)
Dopamine Rat 1 :1 .000 Millipore
transporter (DATL QVIA8369)
p67Phox Mouse 1:500 BD Biosciences
San Jose. CA
(611415)
gp91Phox Mouse 1:500 BD Biosciences
(61091 3)
Alpha—synuclein Rabbit 1 :1 .000 Cell Signaling
(2642)
Dopamine and Rabbit 1:1.000 Cell Signaling
cyclic AMP- (2302)
regulated
phosphoprotein
(DARPP-32)
Parkin Mouse 1 :1 .000 Millipore
(MA85512)
Glyceraldehyde—3— Mouse 1 25,000 Millipore
Phosphate (MAB374)
Dehydrogenase
(GAPDH)
Beta-Iii tubulin Mouse 122,000 Millipore
(MAB1 637)
Synaptosomal- Mouse 1 22,000 Millipore
associated Protein (MAB331)
25 (SNAP-25)
lRDye 800 Goat 1 :5.000-1:20.000 Rockland,
conjugated goat Gilbertsville, PA
anti-rabbit IgG (611-132-122)
ALEXA Fluor 680 Goat 1:5.000-1:20.000 Molecular Probes,
goat anti-mouse Eugene, OR
lgG (A-21057)
lRDye 800 Goat 1:10.000 Rockland
conjugated goat (612-1 32-120)
anti-rat lgG

 

Table 2-3. Primary and secondary antibodies used for Western blotting. The antibody the
protein was directed against, species it was obtained fiom, dilution used, and company

(source) it was obtained from is listed.

 

H. Immunohistochemistry and Stereology Cell Counts
Immunohistochemistry for TH

Hindbrains placed in 4% paraformaldehyde following decapitation or whole
brains perfused with 4% paraformaldehyde were sectioned coronally at 60 pm in a
cryostat at -25 °C. Every third tissue section was stained for TH with a rabbit polyclonal
antibody to TH (1:2,000) (AB152, Chemicon, Temecula, CA). Brain tissue was reacted
with a biotin conjugated goat anti-rabbit secondary antibody (1 :500) (Vector
Laboratories, Burlingame, CA). Secondary antibody was reacted with an avidin biotin
complex using an ABC Vectastain kit (Vector Laboratories, Burlingame, VT), followed
by 3,3'-diaminobenzidine (Sigma-Aldrich) to visualize the staining. All brain tissue
sections were mounted on gelatin coated slides, and dehydrated using a series of steps
through ethanol (70%, 95%, and 100%) and xylene (two steps) each for 10 min prior to

being coverslipped.

Stereological TH Cell Counts

Brain sections stained for TH were visualized on a screen using a 4X objective
and the substantia nigra was delineated with the aid of a mouse atlas using
Stereoinvestigator Software version 6.55 (MicroBrightField, Inc., Williston, VT)
(Franklin and Paxinos, 1996). The first section counted contained TH immunoreactive
cell bodies from the rostral substantia nigra (Bregma -2.80), and subsequent sections
were counted every 180 um following this initial section until TH immunoreactive cells

were no longer visualized.

90

TH positive cells were counted using the unbiased Optical Fractionator technique
with a 20X objective on a Nikon Eclipse 80i microscope and an Optronics Microfire
color CCD camera. This technique allows an unbiased estimation of the number of cells
in a region using a three dimensional probe or optical disector, and a systematic sampling
method or fractionator (Gundersen and J ensen, 1987; Schmitz and Hof, 2005). A (100 x
100 um) counting frame was used and a fraction of the counting frames were sampled in
the delineated region of each section. Mounted section thickness was estimated to be 18
um and 2 pm guard zones were set at the top and bottom of sections, so approximately
78% of a section was counted. A cell was considered to be a TH immunoreactive neuron
if the cytoplasm of the neuronal cell body stained positive for TH, the nucleus of the
neuron could be Clearly visualized, and the cell body was greater then 15 pm in diameter.
Uniform antibody penetration was confirmed by the distribution of immunoreactive cell
counts in the vertical (2) axis. To accurately calculate the number of TH immunoreactive
cells in a unilateral substantia nigra every third section was analyzed (section periodicity
of 3) and 8-9 sections were analyzed through the substantia nigra. The mean coefficient
of error (C. E.) was calculated to be 0.1 or less for each treatment group (Gunderson,

m=l) (Gundersen and Jensen, 1987).

91

l. NADPH Oxidase Activity Assay
Brain Tissue Preparation

NADPH oxidase activity was measured in striatum or ventral midbrain brain
tissue samples. Brain tissue was removed and immediately frozen on dry ice, and
sectioned in a cryostat at -10°C. Two 12 gauge tissue punches of striatum were taken
from 500 uM tissue sections or dissected out for ventral midbrain tissue (Figure 2-8), and
placed in 200 uL of lysis buffer (0.1 M KZPO4, 1 mM phenylrnethanesulfonyl fluoride
(PMSF), 0.002% Triton-X), and sonicated to disrupt cell membranes. Samples were
centrifuged at 12,000 xg for 30 min at 4°C, supernatant was removed and the pellet
resuspended in 150 uL of a reaction buffer (20 mM HEPES, 0.12 M NaCl, 4.6 mM KCl,
2 mM MgSO4, 0.15 mM Na2HPO4, 0.4 M KHzPO4, 5 mM NaHCOg, 5.5 mM Glucose,
1.2 mM CaClz, and pH 7.4), and re-homogenized by sonication (Pagano et al., 1995; Li et
al., 2003). Protein content of samples was determined using a BCA protein assay

described in (Chapter 2, Section G, Brain Tissue Preparation).

NADPH Oxidase Activity Determination

NADPH oxidase activity was measured using 250 ug of protein for each sample
placed in 500 uL of reaction buffer with 1 mM PMSF, 10 ug/mL of aprotinin, 10 ug/mL
of leupeptin. Enzyme activity was measured by incubating protein samples in a water
bath for 10 min at 37°C, 5 uL of 10 mM NADPH and 5 uL of 0.5 mM lucigenin was
added to the sample and incubated for another 10 min in a water bath at 37°C. The 1.5
mL tube containing the sample was placed in a 20/20n luminometer (Turner BioSystems,

Sunnyvale, CA) for 90 s before a total of 10 readings (5 5) each were taken and expressed

92

as relative light units (RLU), 5 uL of the superoxide scavenger (1 M tiron) was added to
the sample to scavenge superoxide and after 5 min another set of 10 readings (5 3) each
were taken. Results were calculated for NADPH oxidase activity of samples by the
difference between the mean of readings 2 through 9 before and after the addition of tiron
was taken, and a mean background subtraction of the reaction buffer with NADPH and
lucigenin in the presence of no protein was performed. This value was multiplied by a
factor of 12 to account for RLU per min (RLU/min) since each reading was taken over a
5 sec period. This value was divided by the total protein (250 ug) contained in each

sample such that NADPH oxidase activity was expressed as RLU/min/ug of protein.

NADPH oxidase activity = 12*(( mean 2—9 pre-tiron — mean 2-9 yst-tironH mean 2-9 background”
250 ug of protein

93

J. Mitochondrial Complex I Assay

Complex I is the first in the series of the mitochondrial respiratory chain that
involves the transfer of electrons from nicotinamide adenine dinucleotide (N ADH) by
oxidation (Greenarnyre etal., 2001). The Complex I assay uses 2,6-diChloroindophenol
(DCIP) as a terminal electron acceptor, where Complex I present in mitochondria isolated
from brain tissue oxidizes NADH, producing electrons which are transferred to
decylubiquinone (a ubiquinone analog electron carrier) that delivers the electrons to
DCIP. This assay measures the reduction of DCIP at 600 nM every min over a 12 min
period. After the fifth reading, rotenone is added to the reaction mixture to inhibit
Complex I activity within the tissue sample. Complex I activity is determined using the

difference in the absorbance decay before and after the addition of rotenone.

Brain Tissue Preparation

Brains were frozen and sectioned at 500 pm in a cryostat and striatum tissue was
dissected from brain sections. Striatum samples were placed in 10 mM Tris buffer (pH
7.8), and homogenized with a Dounce homogenizer. Tissue homogenates were
centrifuged for 10 min at 1000 x g, supernatant (S1) removed, frozen in liquid nitrogen
and thawed in a water bath. The pellet (P1) fi'om this centrifugation was discarded.
Supernatant (Sl) was centrifuged at 14,000 x g for 20 min and the pellet (P2) from this
centrifugation contained mitochondria. The pellet (P2) was resuspended twice in 10 mM
Tris buffer and re-centrifuged each time at 14,000 x g for 20 min to obtain a Clean

mitochondrial fraction. The pellet (PZ) Was resuspended in 100 mM KH2PO4 buffer pH

94

7.8 and the protein content determined using a BCA assay. Samples were stored at -80°C

until time of the assay.

Optimization of Sample Size

The optimal amount of protein needed to measure Complex I activity was
determined by measuring Complex I activity with 0, 2.5, 5, 10, or 15 ug of striatum
protein. Complex I activity measured at the specified protein concentrations produced a
linear standard curve by plotting the difference in the absorbance decay before and after
the addition of rotenone against protein content within samples (Figure 2-11). A total of 5
ug of protein (within the linear range of Complex I activity) was used in all sample assays

measuring Complex I activity.

Complex I Activity Assay

All samples were run in triplicate using 5 ug protein aliquots for each replicate.
Samples were placed in reaction buffer containing 3.5 g/L bovine serum albumin, 25 mM
KHzPO4, 70 uM decyclubiquinone, 60 uM 2,6-dichloroindophenol (DCIP), and 1 uM
antimycin A(Sigma-A1drich) and measured for Complex I activity (J anssen et al., 2007).
Samples were placed in a Beckman DU460 spectrophotometer and NADH substrate
(Sigma-Aldrich) was added in a final concentration of 0.2 uM. Using a kinetics program
absorbance readings were taken over a 12 min period and rotenone was added to each
sample following the 5 min reading for a concentration of 1 uM rotenone. The difference
between the 2° and 5th readings was taken and subtracted from the difference between the

8th and 11th readings, and divided by the 3 min time interval, and then divided by the

95

appropriate amount of protein added to each sample (5 ug), which was reported as

Complex I activity.

96

0.04 -

 

 

 

E R=0.99
.E 5 0.03 -
8 d)
r: ‘c’ 0.02 -
93 3
#5 3 0.01 -
a in
'2 0.00 . r .
0 5 10 15

Protein Content (ug)

Figure 2-11. Plot of striatum brain tissue protein content verses the difference in
absorbance decay before and after Complex I inhibition. Linearity of Complex I activity
is seen across 2.5-15 ug of protein as indicated by the difference in the rate of absorbance
decrease before and after rotenone addition to samples. Data points represent means and
bars one SEM.

97

K. MPP+ Assay

MPP+ is the active metabolite of MPTP taken up into DA neurons by DAT.
MPP+ can be measured in brain regions using liquid Chromatography coupled to mass
spectrometry (LC-MS). LC-MS is a very sensitive and quantitative method of measuring
MPP+ because it uses HPLC to identify how long it adheres to a hydrophobic column
(retention time) and MS to identify molecular fragmentations specific to MPP+. MS
detection of MPP+ is done by providing mobile phase conditions that allow full ionization
of MPP+, desolvation in an atmospheric pressure source for electrospray ionization, and
acceleration into a tandem, i.e. triple stage quadrupole (TSQ), MS detector. The initial
quadrupole radio frequency and direct current voltages are set to filter the mass/charge
ratio specific to MPP+ (i.e. m/z 170.110). This precursor or parent ion continues into a
second stage hexapole to undergo kinetic fragmentation imparted by collisions with argon
gas, followed by mass analysis in the third quadrupole. When the third quadrupole is set
to look only at specific fragments rather than scanning a range, then this is considered
multiple reaction monitoring or selected reaction monitoring detection, depending on the
manufacturer and its software conventions. Charged fragments bombard a conversion
dynode, and the resultant generated electrons are in turn measured by a neighboring
electron multiplier. The electrical signals fiom the electrons are measured as an amplified
voltage, and the corresponding voltages are collected, stored and analyzed using computer
software.

MPP+ fragment ions are m/z 127, 128 and 154, and quantitation is based on the
m/z 170-) 127 fragmentation, whereas the m/z 1709128 and -)154 fragmentations are

used as qualifiers. Where necessary, the three ions are summed for maximum sensitivity.

98

The three ions arise specifically as: m/z 128, loss of CH3N=CH; m/z 127, loss of H fiom
m/z 128; and m/z 154, simultaneous loss of H and CH3 from m/z 170, a loss that may be
specific to methylated quartemary amines, since the related compound paraquat (1,1 ’-
dimethyl-4,4’-dipyridinium) shows similar loss under ESI-MS conditions (Lee et al.,
2004). Fragment identifications are tentative due to their unusual individual natures, but in
any case agree with spectra generated by others under similar conditions (Boismenu et al.,

1996).

Brain Tissue Preparation

Brain tissue was sectioned at 500 uM in a cryostat at -10°C, mounted directly onto
room temperature glass slides, and refrozen on dry ice. Micropunches of the striatum
were sampled for MPP+ determination. An 18 gauge circular punch tool (1 mm inner
diameter) was used to obtain striatum tissue (Bregma +1.42) (Franklin and Paxinos,
1996). Brain tissue punches were placed in 30-50 uL of tissue buffer and centrifuged at
13,000 x g for 1 min to pellet tissue. Tissue was sonicated using three 1 s bursts and
samples centrifuged again at 13,000 x g for l min to pellet brain tissue. Samples were q.s.
to 60 uL with tissue buffer, and brain tissue pellets were placed in 100 uL of 1 N NaOH to
determine protein content using a Lowry protein assay (Lowry et al., 1951). Samples
were centrifuged again at 18,000 x g for 3 min, and then transferred to glass vials for LC-

MS analysis.

99

LC-MS Analysis of MPP+

Brain tissue samples were prepared and 10 uL of each sample was injected onto a
Thermo-Finnigan Surveyor HPLC connected to a Thermo-Finnigan TSQ Quantum ESI-
MS/MS detector (Thenno-Fisher Scientific Inc., Waltham, MA). A series of known
MPP+ standards (MPP+ 0, 0.5, l, 2, 5, 10, 20, 50, and 100 ng/mL) were also measured,
and the peak area of MPP+ standards was plotted against the known concentrations of
individual standards (Figure 2-12). The linear equation of the line resulting from this plot
was used to determine sample MPP+ concentrations by interpolating the peak area of
samples in the equation of the line. The peak areas of standards and samples were
measured using the analytical software Xcalibur (Therrno-Fisher Scientific). Individual
sample concentrations of MPP+ were normalized to protein content of brain tissue
samples and expressed as the concentration of MPP+ (ng) over the weight of brain tissue
(mg). The limit of detection for MPP+ concentrations was approximately 5 ng/mL, thus
any value below 5 ng/mL or approximately 6 rig/mg (assruning a tissue punch protein

concentration of 50 ug) are essentially zero.

100

   
  

2000 -

.3
01
O
O
r

1000 a

 

Peak Area * 1.000

01
O
O O
r

 

l I I I 1

20 40 60 80 100
MPP+(ngImL)

C

Figure 2-12. Standard curve of MPP+ concentrations measured using liquid
chromatography mass spectrometry. MPP+ standards (MPP+ 0, 0.5, 1, 2, 5, 10, 20, 50,
and 100 ng/mL) were measured, and the peak area of MPP+ standards was plotted against
the known concentrations of individual standards.

101

L. Statistical Analysis

Power of studies was set at 0.8 and used to the determine number of animals or
samples required for significant strength. Data analysis between only two sample groups
concerning one variable used a Students T-test to determine if statistical significance was
present. For data analysis in which one variable was investigated with more then two
sample groups a one-way analysis of variance (AN OVA) test was used. Data from
experiments involving more than two sample groups and having two or more variables
were analyzed for statistical significance using a two-way ANOVA by a post hoc Tukey’s
test for multiple comparisons. For all statistical tests an alpha value of p g 0.05 was
deemed statistically significant. All statistical tests used were done using Sigma Stat

Statistical Software (Systat Software, Point Richmond, CA).

Notes

Images in this dissertation are presented in color.

102

Chapter 3: Effects of Sildenafil on Nigrostriatal Dopamine Neurons in a Murine

Model of Parkinson’s Disease

A. Introduction

Neurodegenerative diseases, like PD, progressively worsen in concert with the
inexorable loss of specific neuronal populations. The motor symptoms of PD, slowness
of movement, resting tremor, rigidity, and postural instability, are primarily due to the
loss of the NSDA neurons (F ahn, 2003). Typically PD is not diagnosed until
approximately 40-60% of NSDA neurons are already lost (Dauer and Przedborski, 2003).
L-DOPA or DA agonists are currently used to ameliorate the motor symptoms that occur
due to deficits of striatal DA in patients with PD, but these symptomatic treatments do
not prevent the progressive loss of NDSA neurons or decline of motor function in PD
(Lau and Meredith, 2003). Despite substantial advances in our understanding of
molecular pathogenic mechanisms underlying cell death in PD and other
neurodegenerative diseases, no treatments have proven neuroprotective efficacy in the
clinical setting (Ravina et al., 2003). As such, validating a neuroprotective agent to slow
or halt the progression of PD remains an important goal for therapy development.

Several animal models have been employed to study potential neuroprotective
agents, one of these being the MPTP mmine model of PD (Dauer and Przedborski, 2003).
Various MPTP dosing paradigms have been used to model PD in mice and the chronic
exposure paradigm may be particularly advantageous when screening for neuroprotective
effects of target compounds (Petroske et al., 2001). MPTP readily crosses the blood
brain barrier and is converted to its active metabolite MPP+ by the enzyme MAO B.

MPP+ is a relatively selective NSDA neuronal toxicant and concurrent administration of

w 103

probenecid prevents the loss of MPP+ from the brain and extends its duration of action
(Lau et al., 1990; Tipton and Singer, 1993; Przedborski et al., 2001). The Chronic MPTP
model of PD causes striatal DA depletion and a progressive loss of NSDA neurons over
an extended period of time (Meredith et al., 2002). Taken together, the features of the
Chronic MPTP-probenecid murine model mirror the progressive nature NSDA neuronal
loss in PD and provide an unique model to study potential neuroprotective agents for this
disease (Chan et al., 2007).

Phosphodiesterases are intracellular enzymes that catalyze the breakdown of 3’,
5’-cyclic nucleotides such as cGMP, into inactive 5’-monophosphate nucleotides
(Kulkarni and Patil, 2004). Phosphodiesterases have varying specificities for 3’, 5’-
cyclic nucleotides; PDES is selective for cGMP, whereas others catabolize both cGMP
and cyclic adenosine monophosphate (CAMP) (Corbin and Francis, 1999; Kulkarni and
Patil, 2004). PDES (or its mRNA) is found in specific cell types in several brain regions
including Purkinje cells of the cerebellum, motor and non-motor neurons of the spinal
cord, and in adult stem cells of the subventricular zone (SVZ) (Kotera et al., 2000;
Nakamizo et al., 2003; Shimizu-Albergine et al., 2003; Van Staveren et al., 2003; Wang
et al., 2005). mRNA for PDES has also been found in the olfactory bulb, cortex,
hippocampus, and substantia nigra, but the phenotypes of cells containing this enzyme
are not known (Loughney et al., 1998; Van Staveren et al., 2003).

Sildenafil is a PDE5 inhibitor approved for the treatment of erectile dysfunction
(ED), a common non-motor symptom in PD (Corbin and Francis, 1999; Michelakis et al.,
2000; Kulkarni and Patil, 2004). In addition to its vasodilatory action on penile

microcirculation, Sildenafil also increases cortical concentrations of cGMP and promotes

104

neurogenesis in the SVZ of rats with ischemic stroke as demonstrated by an increase in
the number of bromodeoxyuridine positive cells and immature neurons in the striatum
(Corbin and Francis, 1999; Zhang et al., 2002; Zhang et al., 2006a). The neurogenic
effect of Sildenafil on SVZ cells is associated with cGMP-induced activation of the PI-
3K/Akt pathway. This pathway has also been implicated in the cGMP-mediated survival
of cerebellar granule cells and PC-12 cells (Ciarri et al., 2002; Ha et al., 2003). Increased
intracellular cGMP concentration also inhibits apoptosis of SH-SYSY neuroblastoma
cells (Andoh et al., 2003). Elevations in intracellular levels of CGMP in immune
microglial cells following inhibition of PDES results in a decrease in the release of
various pro-inflammatory cytokines from LPS stimulated microglia suggesting that
Sildenafil could potentially inhibit microglial activation that mediates the oxidative stress-
induced damage to NSDA neurons in mice exposed to MPTP and in neurodegenerative
diseases like PD and Alzheimer’s disease (Paris et al., 1999; Paris et al., 2000; Wu et al.,
2003)

The purpose of these experiments was to evaluate the potential of Sildenafil as a
neuroprotective agent for PD by examining the dose-response effects of three treatment
paradigms of Sildenafil on NSDA neurons in MPTP treated mice. These studies
examined the effects of chronic Sildenafil treatment, initiated before (pm-treatment),
during (concurrent treatment) or after chronic MPTP administration (post-treatment).
Once initiated, chronic Sildenafil administration continued throughout the duration of
these experiments. Endpoints of toxicity included TH protein levels, DA storage and
metabolism in axon terminals of NSDA neurons in the striatum, in addition to the

numbers of NSDA neurons in the substantia nigra.

105

Hypothesis: Sildenafil will attenuate the MPTP induced loss of NSDA neurons when
treatment is initiated before, during, or after the completion of Chronic MPTP treatment.
1) Effectiveness of Sildenafil will be determined by correlating the amount of
free Sildenafil in plasma with concentrations of cGMP within the hindbrain
following Sildenafil injection.
2) The integrity of NSDA axonal terminals will be evaluated by measuring the
concentrations of DA and TH protein content in the striatum following Chronic
MPTP and sildenifil treatment.
3) NSDA neuronal cell body integrity will be determined by counting the number
of TH immunoreactive cells in the substantia nigra after Chronic MPTP

treatment and Sildenafil pre-treatment.

106

B. Materials and Methods

Male C57BL/6 mice (Jackson Labs) aged 8-10 weeks were used. Mice that
received MPTP were housed separately from saline treated control mice. Mice were
injected with either 0.9% NaCl or MPTP (20 mg/kg, SC) and probenecid (250 mg/kg, i.p.
using a Chronic treatment regimen every 3.5 days for 35 days as previously described
(Chapter 2, Section B Drugs). Sildenafil Citrate (5 or 10 mg/kg) or 0.9% NaCl were
administered twice daily to mice via s.c. injection as previously described (Chapter 2,
Section B Drugs).

Mice were killed by decapitation 21 days following the last MPTP injection. The
forebrain was dissected at the coronal plane midway through the hypothalamus. The
forebrain was prepared for neurochemical and Western blotting analysis of TH protein
content as previously described (Chapter 2, Section D Assay of Amines, Amine
Metabolites, and Apocynin in Brain Tissue and Section G Western Blot Analysis). The
remaining hindbrain was dissected at the mid-saggital plane and half of the hindbrain was
immersion fixed in 4% paraformaldehyde and stored at 4°C for TH staining and the other
frozen on dry ice for analysis of cGMP content.

Unilateral hindbrains were sectioned at 60 um using the MultiblockTM processing
method and cells stained for TH by Neuroscience Associates (Knoxville, TN). Briefly,
every other coronal tissue section was stained using rabbit anti-TH 1:1,500 (Pelfreez
Biologicals, Rogers, AR). Primary antibody was bound with the appropriate biotin
conjugated secondary antibody, followed by an avidin biotinylated enzyme complex
(Vector Laboratories, Burlingame, VT). Bound peroxidase was visualized using 0.05%

3-3'-diaminobenzidine tetrahydrochloride with 0.01% hydrogen peroxide. The number of

107

immunoreactive TH cells was then determined using an unbiased stereological method as
previously described (Chapter 2, Section H, Immunohistochemistry and Stereology Cell
Counts). Brain tissue was also stained for the neuroblast marker doublecortin to
determine if neurogenesis occurred within the substantia nigra, and doublecortin positive
cells observed in the hippocampus were used as a positive control.

The concentrations of free Sildenafil in plasma was determined 30 nrin after a
single (5 or 10 mg/kg s.c.) Sildenafil injection as previously described (Chapter 2, Section
F, Plasma Sildenafil Determination). The concentrations of cGMP were measured in
hindbrains 30 min following Sildenafil administration as previously described (Chapter 2,

Section E, cGMP assay).

108

C. Results

Blood levels of free Sildenafil in mice 30 min following Sildenafil administration
are shown in (Figure 3-1). Subcutaneous administration of Sildenafil resulted in a mean
of 40-60 nM concentrations of free Sildenafil in plasma following the 5 mg/kg dose, and
approximately 80-85 nM concentrations following the 10 mg/kg Sildenafil dose.
Sildenafil was not detected in the plasma of saline treated mice. cGMP levels were
increased in the brain with either 5 or 10 mg/kg sildenafil treatment 30 min following
administration (Figure 3-1). Treatment with MPTP did not affect levels of Sildenafil in
plasma or alter Sildenafil-induced increases in brain cGMP levels.

Chronic exposure to MPTP produced a marked decrease in DA concentrations in
the striatum (Figures 3-2, 3-3, & 3-4). There was a corresponding, but less marked,
decrease in striatal levels of TH protein (Figure 3-5). A decrease in TH immunoreactive
neurons (Table 3-1, Figure 3-6) was observed in the substantia nigra following chronic
MPTP. The loss of TH-immunoreactive cells parallels the loss of Nissl stained cells in
previous studies (Petroske et al., 2001; Behrouz et al., 2007), indicating the loss of TH-
immunoreactive cells is from cell death and not merely Changes in TH protein expression.
Striatal DOPAC concentrations were reduced (Figures 3-2, 3-3, & 3-4) and the ratio of
DOPAC to DA increased (Figures 3-2, 3-3, & 3-4) following chronic MPTP treatment.

Sildenafil did not alter basal levels of striatal DA or DOPAC, or the ratio of
DOPAC to DA (Figures 3-2, 3-3, & 3-4) at doses of 5 or 10 mg given twice daily.
Similarly, Sildenafil did not alter the levels of striatal TH protein content (Figure 3-5) or
the numbers of TH immunoreactive neurons found in the substantia nigra (Table 3-1).

Chronic MPTP exposure decreased striatal DA and DOPAC concentrations, and

109

increased the ratio of DOPAC to DA to a similar extent in animals treated with 5 or 10
mg/kg of Sildenafil, regardless of whether Sildenafil treatment was initiated before
(Figure 3-2), concurrently or after (Figure 3-3 & 3-4) MPTP administration. Similarly,
MPTP-induced decreases in striatal TH protein content (Figure 3-5) and numbers of TH
immunoreactive cells in the substantia nigra (Table 3-1, Figure 3-6) were not affected by
Sildenafil pre—treatment, concurrent treatment, or post-treatment paradigms. Doublecortin
staining used to indicate neurogenesis within the substantia nigra was not observed,

whereas doublecortin positive control cells within the hippocampus were seen.

110

?

Plasma Sildenafil Levels

 

 

 

‘2‘ .
5 100
To 80 -
c
o
2 60
w r
“i: 40 ~
a
g 20 4
'5 ND. no.
0 .-
0 5 1o 0 5 10

Control MPTP
Sildenafil dose (mg/kg)

Hindbrain cGMP Content

N
o
n

 

d
01
n

 

 

.0
OI
L

 

cGMP (fmollug of protein)

   

 

 

 

 

__|

0 5 10 0 5 10
Control MPTP

Sildenafil dose (mg/kg)

Figure 3-1. Plasma free Sildenafil (Panel A) and brain cGMP levels (Panel B) 30 min
after Sildenafil administration. Mice were injected with either 5 or 10 mg/kg Sildenafil, or
0.9% saline vehicle. Columns represent the means and vertical lines one standard error
of the mean (SEM.), n=4-8. Plasma levels of fi'ee Sildenafil were not detectable (N .D.)
in vehicle controls, but were measurable following 5 and 10 mg/kg Sildenafil treatment in
both control and Chronic MPTP treated mice. Hindbrain cGMP concentrations were
increased following 5 and 10 mg/kg Sildenafil administration in saline and MPTP treated

mice.

 

.0
o
I

111

Sildenafil Pro-Treatment
250 a

 

 

 

 

 

 

 

 

 

 

 

 

DControl
a 200 - IMPTP
E .—l—
a 150 - P'- .1.
E.
< 100 -
o
50 - * * *
o g I -_
O 5 1O
Sildenafil dose (mg/kg)
B.
A 40 .
g, 354 DControl
~ IMPTP
g 301
O
<
a.
O
D
0 5 10
Sildenafil dose (mg/kg)
C.

 

 

Sildenafil dose (mg/kg)

Figure 3-2. Effects of Chronic MPTP administration (20 mg/kg s.c., every 3.5 days for 35
days) on DA and DOPAC concentrations, and the DOPAC/DA in the striatum of mice
pre-treated with Sildenafil. Mice were treated with either saline vehicle or Sildenafil (5 or
10 mg/kg, s.c., twice daily) beginning 3 days before saline vehicle or MPTP
administration and continuing until the day of decapitation. Columns represent means
and vertical lines one standard error of the mean SEM. * P<0.05, n=4-9. DA and
DOPAC concentrations (Panels A & B) significantly (*) decreased following chronic
MPTP treatment, and were not affected by Sildenafil treatment. The DOPAC/DA ratio
(Panel C) increased significantly (*) increased with MPTP treatment, but was not

affected by sildenafil treatment .

112

Sildenafil Concurrent Treatment

 

 

 

 

 

 

 

 

 

 

 

 

 

300 ' DControl
g 250 . IMPTP
a, 200- F“
f 150 -
50 -
0 l I___-_
o 5 10
Sildenafil dose (mg/kg)
2g ' DC trl
A . on 0
g 40 . _L i IMPTP
‘ 35 r
E 30 « -‘-
u 25 '
< 20 r * *
8 15 r *
10 a
o
5. I L 1.
o ___ _ _
0 5 10
Sildenafil dose (mg/k9)
0.6 - DControi
g 0.5 , IMPTP
2 0.4 - *
3 0.3 -
o 0.2 “
0.1 -
0.0 -

 

0

 

5

 

10

Sildenafil dose (mg/kg)

113

Figure 3-3. Effects of Chronic MPTP administration (20 mg/kg s.c., every 3.5 days for 35
days) on DA and DOPAC concentrations, and the DOPAC/DA in the striatum of
concurrent Sildenafil treated mice. Mice were treated with saline vehicle or Sildenafil (5
or 10 mg/kg, s.c., twice daily) beginning 3 days after saline vehicle or MPTP
administration began (Concurrent Treatment) and continuing until the day of
decapitation. Columns represent means, vertical lines one standard error of the mean
SEM.. *, P<0.05, n=5-9. DA and DOPAC concentrations (Panels A & B) significantly
(*) decreased with Chronic MPTP treatment, but were not affected by Sildenafil treatment.
The DOPAC/DA ratio (Panel C) significantly (*) increased with MPTP treatment, but
was not affected by Sildenafil treatment.

Sildneafil Post-Treatment

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

300 _ DCOMI’OI
E, 250 .. IMPTP
a 200 - ""1
f 150 4
50 -
0 1 L__-_
0 5 10
Sildenafil dose (mg/kg)
B.
50
g l DControl
40 IMPTP
r.» j ”5 FL 4.
~v 30
2
5 ’°‘ .
o 10 - * *
o __I__._I_
0 5 10
Sildenafil dose (mg/kg)
C.
0.6 -

DControl
< 0-5 ‘ IMPTP
Q
0
.<
n.

O
D

 

 

0 5 10

Sildenafil dose (mg/kg)

Figure 3-4. Effects of Chronic MPTP administration (20 mg/kg s.c., every 3.5 days for 35
days) on DA and DOPAC concentrations, and the DOPAC/DA in the striatum of post-
treatrnent Sildenafil treated mice. Mice were treated with saline vehicle or Sildenafil (5 or
10 mg/kg, s.c., twice daily) beginning 3 days after saline vehicle or MPTP administration
ended (Post-Treatment) and continuing until the day of decapitation. Columns represent
means, vertical lines one standard error of the mean SEM.. *, P<0.05, n=5-9. DA and
DOPAC concentrations (Panels A & B) significantly (*) decreased with Chronic MPTP
treatment, but were not affected by Sildenafil treatment. The DOPAC/DA ratio (Panel C)
significantly (*) increased with MPTP treatment, but was not affected by Sildenafil

treatment.

114

Sildenafil Pre-Treatment

 

 

 

 

 

 

 

 

 

 

CiControl
g 301 IMPTP
E 601 r'L -’- 41
E 40-
: l I l
E o
0 5 10

Sildenafil dose (mg/kg)

Sildenafil Sildenafil
Sal 5 mg tome/k9

Sal MPTP Sal MPTP Sal MPTP

BetailiTubulin—> ~u— — ~ I. —
50kDa

Figure 3-5. Effect of Chronic MPTP administration (20 mg/kg s.c., every 3.5 days for 35
days) on TH protein content in the striatum of mice pre-treated with Sildenafil. Mice
were treated with either saline vehicle or Sildenafil (5 or 10 mg/kg, s.c., twice daily)
beginning 3 days before saline vehicle or MPTP administration and continuing until the
day of decapitation. Columns represent means, vertical lines one standard error of the
mean S.E.M., *, P<0.05, n=6-8. TH protein content (Panel A) significantly (*) decreased
with Chronic MPTP treatment, but was not affected by Sildenafil treatment.
Representative Western blots of TH and Beta III Tubulin (Panel B) in the striatal
cytosolic fraction.

115

 

Figure 3-6. Effects of chronic MPTP administration (20 mg/kg s.c., every 3.5 days for 35
days) on TH immunoreactive cells in the substantia nigra. TH immunoreactive cell
bodies and fibers are stained brown and bar = 500 um. Representative pictures from
selected treatment conditions are shown. Saline control and saline vehicle treated (Panel
A), saline and 10 mg/kg Sildenafil Pre-Treatment (Panel B), MPTP and saline vehicle
treated (Panel C), MPTP and 10 mg/kg Sildenafil Pre-Treatment (Panel D), MPTP and 10
mg/kg Sildenafil Concurrent Treatment (Panel B), MPTP and 10 mg/kg Sildenafil Post-
Treatrnent (Panel F).

116

Table 3-1. Numbers of TH Immunoreactive Cells in the unilateral Substantia Nigra Pars
Compacta of Sildenafil and/or MPTP treated Mice

 

 

 

Treatment Mean :h S.E.M. % Loss C.E.
SalineNehicle 3094 :h 253 - 0.08
Saline/10 mg/kg Sildenafil Pre-Treatment 3254 :l: 182 - 0.07
MPTPNehicle 2185 d: 113* 29 0.09
MPTP/ 10 mg/kg Sildenafil Pre-treatment 2150 i 242* 31 0.09
MPTP/10 mg/kg Sildenafil Concurrent 1509 d: 106* 49 0.10
Treatment

MPTP/10 mg/kg Sildenafil Post-Treatment 2128 :l: 216* 31 0.09

 

Table 3-1. Data are presented as the mean i the S.E.M. and percent loss of TH
immunoreactive cell bodies of the saline/vehicle treated control. * Values that are
significantly different (P< 0.05) from control. Neurons immunoreactive for TH were
counted using unbiased stereological counts obtained using an optical fractionator
technique to control for variations in neuronal size and volume of the region being
analyzed. Data is reported as an average number of total TH positive neurons per
unilateral substantia nigra.

117

D. Discussion

The results of this study reveal that chronic administration of MPTP causes a
significant decrease in striatal DA and DOPAC concentrations and TH protein content, as
well as a significant loss of TH positive cells in the substantia nigra. A decrease in the
concentration of DA in the striatum of MPTP treated mice reflects a loss in the density of
NSDA neuronal terminals that is supported by a decrease in striatal TH protein content.
Sildenafil treatment did not prevent the MPTP-induced loss of DA, DOPAC, or TH
content in the striatum, or alter basal levels of these parameters. The compensatory
increase in firnction of remaining NSDA neurons, as indicated by the increased
DOPAC/DA ratio with MPTP treatment, was also not altered with Sildenafil treatment.
Therefore, Sildenafil did not affect the function or compensatory increase in activity of
remaining NSDA neurons after MPTP treatment. A significant decrease in the number of
TH immunoreactive cells in the substantia nigra of MPTP treated mice indicated a loss of
NSDA neuronal cell bodies in this region, which was not affected by Sildenafil treatment.

The data presented demonstrates the reliable and reproducible effects of Chronic
MPTP exposure on clearly defined endpoints of neurodegeneration, thus underscoring the
utility of this model (Lau et al., 1990; Petroske et al., 2001; Drolet et al., 2004). The
Chronic and progressive nature of the MPTP-induced disruption of NSDA neurons
observed recapitulates that of PD, which provides distinct advantages over acute dosing
models for the study of potential neuroprotective agents. Several different MPTP
treatment paradigms have been used to study PD in mice each with a different extent of
loss of striatal DA and DOPAC concentrations and NSDA neuronal cell bodies in the

substantia nigra (Lau et al., 1990; Przedborski et al., 2001; Schmidt and F erger, 2001).

118

MPTP is converted to its toxic metabolite MPP+ which inhibits Complex I of the
mitochondrial respiratory chain, causes ATP depletion, oxidative stress, and necrotic and
apoptotic loss of NSDA neurons (Tipton and Singer, 1993; Dauer and Przedborski, 2003;
Tretter et al., 2004; Novikova et al., 2006). However, the depletion of striatal DA and
DOPAC concentrations and TH immunoreactivity in the striatum and substantia nigra
have been demonstrated to be partially or completely reversible using some of these other
MPTP treatment paradigms, suggesting that loss of neurons may not occur using these
treatment paradigms (Lau et al., 1990; Petroske et al., 2001; Schmidt and F erger, 2001).
In contrast, the Chronic MPTP paradigm used in this study causes a sustained loss of
NSDA neurons, which allows a comparison of pre-treatment, concurrent treatment and
post-treatment paradigms when screening for neuroprotective effects of lead compounds
(Lau et al., 1990; Petroske et al., 2001).

Several lines of evidence suggest that Sildenafil could be a neuroprotective agent
for PD. Indeed, due to its ability to block PDES and increase cGMP concentrations in the
brain, Sildenafil promotes neurogenesis of SVZ cells in rats with ischemic stroke and
inhibits the release of inflammatory cytokines from microglial cells (Paris et al., 1999;
Paris et al., 2000; Zhang et al., 2002; Zhang et al., 2006a). Serum deprived human
neurotrophic SH-SY5Y cells with elevated cGMP concentrations or following treatment
with a CGMP analogue activate intracellular protein kinase G (PKG) thereby inducing the
anti-apoptotic protein BCL-2, which has been shown to attenuate MPT P induced
neuronal toxicity when overexpressed in mice (Yang et al., 1998; Andoh et al., 2000;
Andoh et al., 2003). When these cells were treated with MPP+, they showed increased

resistance to MPP+ toxicity thus implicating a neuroprotective mechanism via elevation

119

of cGMP concentrations (Andoh et al., 2000; Andoh et al., 2002; Andoh et al., 2003). A
cGMP analogue, 8-Br-CGMP, that can activate PKG was shown to decrease 6-OHDA-
induced PC-12 cell apoptosis, by suppressing a mitochondrial apoptosis signal which is
associated with 6-OHDA cell death (Ha et al., 2003). 8-Br-CGMP also decreases NGF
withdrawal- and B-amyloid-induced cell death in PC—12 cells (Wirtz-Brugger and
Giovanni, 2000). Another cGMP analogue, dibutyryl cGMP, as well as a nonspecific
PDE inhibitor, IBMX, was shown to elevate the release of brain-derived neurotrophic
factor (BDNF) implicated in increased survival of NSDA neurons in a DA cell line,
suggesting a that increased cGMP was neuroprotective (Chun et al., 2000). Taken
together, this evidence suggests that Sildenafil-induced elevations in cGMP
concentrations could potentially be neuroprotective.

To determine if Sildenafil was neuroprotective in the chronic MPTP model of PD
three different treatment paradigms were used. Sildenafil administration was initiated
either 3 days before (pre-treatrnent), three days following (concurrent treatment) the
initiation of MPTP treatment, or 3 days following the last injection of MPTP (post-
treatment) and continued until sacrifice. These various paradigms permitted the
evaluation of Sildenafil to prevent NSDA neuronal death before, during, or after the
initiation of MPTP exposure. The post-treatment paradigm is particularly compelling,
since it recapitulates the most likely scenario in which a neuroprotective drug would be
employed in patients with PD; i.e., in the absence of a valid biomarker of PD to detect
pre-Clinical disease, most patients could only be administered a neuroprotective therapy

after the disease process has been initiated.

120

The results from these experiments, however, do not support a neuroprotective
effect of Sildenafil on NSDA neurons in the chronic MPTP model. The administration of
Sildenafil was sufficient to produce an increase in brain cGMP concentrations, which is
consistent with previous studies that found systemic administration of similar or lower
doses of Sildenafil increased rodent brain cGMP content (Zhang et al., 2002). That said,
low basal PDES expression in the NSDA neurons could limit the Sildenafil-induced
increase of cGMP concentrations in NSDA neurons, which could attenuate any potential
neuroprotective effects of Sildenafil in the chronic MPTP model (Loughney et al., 1998;
Wykes et al., 2002; Van Staveren et al., 2003). In addition, it should be noted that
Sildenafil has a relatively short-half life and PDE-5 inhibitors with a longer half-life could
provide a more sustained Change in cGMP levels. Finally, repeated administration of
Sildenafil could have down-regulated PDE-5 expression and limited the long-term
efficacy of this compound.

Prolonged exposure to repeated Sildenafil treatment alone did not adversely affect
the integrity of NDSA neurons, both under basal conditions and in the context of NSDA
damage. The MPTP-induced decrease in striatal DA concentration, TH protein content,
and the loss of TH immunoreactive cells in the substantia nigra was similar in vehicle and
Sildenafil treated mice. Therefore Sildenafil did not affect MPTP induced
neurodegeneration of NSDA neurons that occurs with chronic MPTP treatment,
suggesting that the use of Sildenafil to treat PD patients with ED would not be
deleterious. ED is a common problem in men with PD and its incidence is significantly
greater in these men than age matched controls. The frequency of ED in PD is not

surprising since the mesocortical and mesolimbic DA pathways affected in PD partly

121

mediate the maintenance of penile erection (Magerkurth et al., 2005; Papatsoris et al.,
2006). Sildenafil administered to men with PD and other neurodegenerative diseases has
been shown to improve sexual function with little or no short-term adverse side effects
(Zesiewicz et al., 2000; Hussain et al., 2001). The results from the present study are in
agreement with the conclusion that the repeated use of Sildenafil in PD patients would not
accelerate the loss of NSDA neurons or alter their function, even in the presence of
ongoing neurodegeneration. As such, Sildenafil administration would not be expected to
adversely affect disease progression or motor function in patients with PD.

In summary, the results from this study indicate that Sildenafil is not
neuroprotective in a Chronic MPTP murine model of PD when Sildenafil treatment is
initiated prior to, during or after neurotoxic insult. Our results also show no deleterious
effects of Sildenafil in the chronic MPTP murine model of PD. Accordingly, Sildenafil

could be considered a safe treatment for ED in men with PD.

122

Chapter 4: Apocynin as a Neuroprotective Agent in the MPTP Model of PD

A. Introduction

NADPH oxidase activation contributes to microglial induced loss of NSDA
neurons through the production of superoxide. Although superoxide is not detrimental
itself, it induces the production of secondary reactive oxygen species such as hydrogen
peroxide and hydroxyl radical that promote micro glial proliferation, and induce the
synthesis and release of pro-inflammatory cytokines and reactive nitrogen species
(J ekabsone etal., 2006; Mander et al., 2006). NADPH oxidase is also implicated in the
pathogenesis of NSDA neuronal cell loss when microglia are either directly or indirectly
activated in the substantia nigra (Wu et al., 2003; Qin let al., 2004; Purisai et al., 2007;
Rodriguez-Pallares et al., 2007). MPTP treatment also induces the activation of NADPH
oxidase, and mice lacking the key functional subunit gp91Phox are partially resistant to
MPTP induced loss of NSDA neurons (Wu et al., 2003). These findings indicate the
importance of NADPH oxidase activation to microglial induced loss of NSDA neurons.

A potential way to reduce NADPH oxidase activation and thereby attenuate
microglial mediated loss of NSDA neurons is through the use of an inhibitor of this
enzyme such as apocynin (acetovanillone, 4-hydroxy-3-methoxyacetophonone).
Apocynin is isolated from the roots of Apocynum cannabinum or Canadian hemp (F elter
and Lloyd, 1898). The roots of Apocynum cannabinum were used by native Americans
for its various medicinal purposes including use as a laxative, anti-rheumatoid agent, and
for coughing and asthma (Moerman, 1998). The active compound can also be isolated

from the roots of the Himalayan plant Picrorhiza kurroa which is used for its anti-

123

inflammatory properties in the Himalayan region (Anonymous, 2001). Apocynin inhibits
NADPH oxidase activation by preventing translocation of the cytosolic subunits p47Phox
and p67Phox to the plasma membrane (Stolk et al., 1994; Barbieri et al., 2004). It has
been suggested that apocynin itself does not inhibit the enzyme but rather one of its
metabolites is responsible for this action (Muller et al., 1999; Ximenes et al., 2007). One
possible mechanism is the oxidation of apocynin by cellular peroxidases, which produces
apocynin radicals that dimerize forming diapocynin. Diapocynin may be the primary
compound responsible for the inhibition of p47Phox translocation to the membrane
(Ximenes et al., 2007).

There are several potential neuroprotective mechanisms of microglial derived
NADPH oxidase inhibition using apocynin. Inhibition of hydrogen peroxide formation
from NADPH oxidase derived superoxide induces microglial cell proliferation and may
attenuate the micro gliosis associated with the loss of NSDA neurons (Mander et al.,
2006). Apocynin inhibition of NADPH oxidase derived superoxide production may also
decrease iNOS induction and consequent nitric oxide and peroxynitrite formation as
demonstrated in vitro (Zhou et al., 1999; Lomnitski et al., 2000; Muij sers et al., 2000).
Metabolites of apocynin are also implicated in inhibiting the migration of cells (Muller et
al., 1999; Klees et al., 2006), which may be beneficial in inhibiting microglial invasion of
the site of inflammation.

Apocynin is a lipophilic molecule which readily crosses the blood brain barrier
and therefore is an ideal candidate to inhibit NADPH oxidase within the brain following
peripheral administration. Apocynin inhibits microglial NADPH oxidase activation in a

rodent model of stroke and reduces the number of activated microglia and degenerating

124

cortical neurons suggesting a potential to reduce microglial activation and loss of NSDA
neurons (Wang et al., 2006). Apocynin also reduces the loss of NSDA neuronal cell
bodies and axon terminals in the 6-OHDA model of PD in rats (Rey et al., 2006),
revealing a potential for it to attenuate or inhibit the loss of NSDA neurons in the MPT P

model of PD.

125

Hypothesis #1: Apocynin rapidly crosses the blood brain barrier following systemic

administration.

leis hypothesis will be tested by:
1) Measuring the concentration of apocynin in the striatum using HPLC-EC to
establish the pharmacokinetics of the drug including how rapidly it crosses the
blood brain barrier and how long it remains within the brain.
2) Investigating the potential effects on dopaminergic neuronal activity by
determining the concentrations of DA and DOPAC in the striatum following
apocynin treatment. ’

Hypothesis #2: MPTP will induce activation of NADPH oxidase that will contribute to

the loss of NSDA neuronal terminals, and thus be attenuated with apocynin treatment.

This hypothesis will be tested by:
1) Measuring the concentrations of DA in the striatum following MPTP
treatment in the presence of apocynin to determine if NSDA axonal terminal
loss reflected by reduced DA concentrations in the striatum is attenuated with
apocynin treatment.
2) Determining the activity of NSDA neurons by measuring the concentrations of
DOPAC and the DOPAC/DA ratio within the striatum following MPTP with and
without apocynin treatment.
3) Evaluating NADPH oxidase activation in the Striatum and ventral
midbrain following MPTP with and without apocynin treatment by determining

the amount of membrane bound p67Phox protein, the protein content of the

126

NADPH oxidase membrane subunit gp91Phox, and the activity of NADPH
oxidase.
For comparison, the effects of MPTP and apocynin treatment on MLDA, TIDA,

and serotoninergic neuronal integrity and activity will also be determined.

127

B. Materials and Methods

Male C57BL/6 mice (Jackson Labs), age 8-10 weeks were used in all experiments
in which mice were treated with MPTP. B6-129X/C57BL/6 crossed mice obtained from
a colony maintained by Drs. John Goudreau and Keith Lookingland were used for
apocynin time course and dose response experiments.

Mice used in apocynin pharmacokinetic experiments were injected i.p. with
apocynin (30, 100, or 300 mg/kg) at various time points prior to decapitation. Brains
were removed and used for analysis of neurochemical and apocynin concentrations which
were measured in the striatum using HPLC-EC as previously described (Chapter 2,
Section D, Assay of Amines, Amine Metabolites, and Apocynin in Brain Tissue).

MAO B activity was measured in the presence of apocynin in vitro to determine if
apocynin inhibits activity of this enzyme. MAO B was added to a reaction mixture of
DA (5 uM) and apocynin (1, 10, or 100 uM) and stopped I h later. The concentrations of
DA and its MAO B metabolite DOPAL were measured by HPLC-EC in reaction samples
as previously described (Chapter 2, Section D, Assay of Amines, Amine Metabolites, and
Apocynin in Brain Tissue) (Legros et al., 2004).

MPTP was administered by one of two paradigms: sub-chronic MPT P (10 mg/kg,
s.c. once a day for 5 days) or repeated acute MPTP treatment (16 mg/kg given i.p. once
every 2 h over a 6 h period for a total of 4 injections; see Chapter 2, Table 2-2). Mice
were killed either 3 days following the last MPTP injection for sub-chronic MPTP
treatment or 2 days following the last MPTP injection for repeated acute MPTP
treatment, and brain tissue was used for neurochemical, Western blotting, or NADPH

oxidase activity assay.

128

The sub-Chronic MPTP model was used in the first study since it induces
microglial activation but does not cause as severe of a loss of NSDA axon terminals as
the repeated acute MPTP model. If apocynin was neuroprotective this may be more
apparent in the sub-Chronic MPTP model. However, lack of an observed neuroprotective
effect in the sub-chronic model led to the use of the repeated acute MPTP model with the
thought that perhaps a greater microglial reaction was needed to observe a potential a
neuroprotective effect of apocynin. Apocynin (100 mg/kg, i.p.) or vehicle administration
for sub-Chronic MPTP treated mice was administered every 12 h beginning 2 days prior
to the beginning of MPTP treatment up to the day of decapitation. Vehicle or apocynin
(250 mg/kg per day) was administered via osmotic mini-pump beginning two days prior
to repeated acute MPTP treatment and continuing until decapitation as previously
described (Chapter 2 Section B, Drugs).

Brain tissue from these experiments was processed for neurochemical analysis of
DA, DOPAC, HV A, SHIAA, and 5HT concentrations in select brain regions as
previously described (Chapter 2 Section D, Assay of Amines, Amine Metabolites, and
Apocynin in Brain Tissue). The protein content of TH, p67Phox, and gp91Phox was
measured in the striatum or ventral midbrain as previously described (Chapter 2, Section
G, Western Blot Analysis). The activity of NADPH oxidase was measured in the
striatum and ventral midbrain following repeated acute MPTP treatment as previously

described (Chapter 2, Section I, NADPH Oxidase Assay).

129

C. Results
Pharmacokinetics of Apocynin in the Brain

The concentrations of apocynin in the striatum were measured using HPLC-EC at
several time points (2.5, 5, 10, 20, and 30 min; Figure 4-1, Panel A) or (2.5, 30, 120, 240,
and 480 min; Figure 4-1, Panel B) following administration of apocynin (100 mg/kg i.p.)
to determine the length of time apocynin remained in the brain. Apocynin concentrations
in the striatum were measurable at the first 2.5 min time point. However, the drug was
rapidly eliminated from the brain essentially being undetectable by 20 min following
administration indicating that apocynin has a half-life of only a few minutes (Figure 4—1)
(unpublished results of Lookingland, Goudreau, and Pappas). The concentration of
apocynin was also measured in the striatum at incremental dosages (0, 30, 100, or 300
mg/kg) at the 2.5 min time point to determine if apocynin crossed the blood brain barrier
in a dose dependent manner. The concentrations of apocynin in the striatum revealed that
apocynin could be measured in the brain in a dose dependent manner, since the dosage of
apocynin formed a linear relationship with the amount of the drug measured in the
striatum (Figure 4-1; Panel C). These results indicated that apocynin is a lipophilic
compound that readily crossed the blood brain barrier, but was quickly eliminated from
the brain possibly due to metabolism or redistribution of apocynin to other tissues.

The concentrations of DA and its metabolite DOPAC were measured in the
striatum at 2.5, 30, 120, 240, and 480 min following apocynin (100 mg/kg, i.p.)
administration to determine if apocynin altered the activity of NSDA neurons. As shown
in Figure 4-2 (Panels A & B) DA concentrations were not altered compared to vehicle

treated mice, but DOPAC concentrations were significantly decreased at 2.5 min

130

following apocynin treatment. DOPAC concentrations returned to vehicle control
concentrations by 30 min following apocynin administration. The DOPAC/DA ratio was
also decreased at 2.5 min due to reduced DOPAC concentrations at that time point
indicating a decrease in NSDA neuronal activity (Figure 4-2; Panel C). DA and DOPAC
concentrations were also measured in the striatum of vehicle and apocynin (30, 100, or
300 mg/kg, i.p.) treated mice at 2.5 min to determine if apocynin effected the activity of
NSDA neurons in a dose dependent manner. A shown in Figure 4-3 (Panels A & B) the
concentrations of DA were not altered with apocynin treatment, but DOPAC
concentrations were significantly decreased in a dose dependent manner with apocynin
treatment. The decrease in DOPAC concentrations in the striatum following apocynin
treatment was associated with a significant decrease in the DOPAC/DA ratio in the
striatum (Figure 4-3; Panel C). The decrease in DOPAC following apocynin could
reflect a Change in NSDA neuronal activity or apocynin inhibition of MAO B
metabolism.

Apocynin inhibition of MAO B was tested by an in vitro cell free experiment
measuring the activity of MAO B in the presence or absence of apocynin (Legros et al.,
2004). MAO B activity was measured over time (15, 30, 45, and 60 min) initially to
determine an ideal time point that MAO B metabolism of DA to DOPAL could be
measured. As shown in Figure 4-4 (Panel A), the reaction over time produced a linear
decrease in DA concentrations and a linear increase in DOPAL concentrations, and based
on these results a 60 min time point was Chosen to terminate the reaction in the presence

of apocynin.

131

As shown in Figure 4-4 (Panels B & C), the synthesis of DOPAL was measured
in the presence of various concentrations of apocynin (1, 10, or 100 uM). Apocynin
treatment alone did not alter the concentrations of DA within the reaction mixture in the
absence of MAO B. The addition of MAO B to the reaction mixture of DA and apocynin
(l, 10, or 100 uM) did not alter the synthesis of DOPAL from DA compared to samples
only containing DA and MAO B at 1 h following the initiation of the reaction. The
results indicated that apocynin likely does not alter metabolism of DA to DOPAC by

MAO B in the brain.

132

 

 

 

 

 

Apocynin
$11200 -
3,1000 4
E. 800 .
-§ 600 -
§ 400 -
2- 200 -
0 -
0 2.5 5 1o 20 30
Time (min)
B.
Apocynin
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g 8001
c 600 -
g 400 «
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C.
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0 50 100 150 200 250 300
Apocynin Dose (mg/kg)

Figure 4-1. Measurement of apocynin in the brain using HPLC-EC. Apocynin was
measured in the striatum of mice at 2.5, 5, 10, 20, and 30 min (Panel A) or 2.5, 30, 120,
240, 480 min (Panel B) following an i.p. injection of 100 mg/kg apocynin, vehicle treated
mice were considered the 0 time controls were injected with vehicle and killed 2.5 min
later, n=4-8. Dose dependent increase of apocynin in the striatum following vehicle, 30,
100, or 300 mg/kg apocynin i.p. injection given 2.5 min prior to decapitation (Panel C),
n=4. Data points represent means and bars one SEM.

133

DA (nglmg) DOPAC (nglmg)

DOPACI DA

 

 

 

DOPAC

20

10

5

0| 1 I I I I I

0 2.5 30 120 240 480

Time (min)
DA

350

300

250 W

200

150

100

50

0| I I I I I I

0 2.5 30 120 240 480

Time (min)
DOPAC/DA

0.10

0.08

0.06

0.04 W

0.02

0.00 I I I I I I l

0 2.5 30 120 240 480
Time (min)

Figure 4-2. Apocynin decreases the concentrations of DOPAC and the DOPAC/DA ratio
in the striatum. Mice were injected with apocynin 100 mg/kg, i.p. and decapitated 2.5,
30, 120, 240, or 480 min later, vehicle treated mice were considered the 0 time controls
were injected with vehicle and killed 2.5 min later, n=7-8. The concentrations of
DOPAC (Panel A) and DA (Panel B), and the DOPAC/DA (Panel C). Data points
represent means and bars one SEM. *Significantly less DOPAC or the DOPAC/DA ratio
compared to vehicle treated mice.

134

 

 

 

 

 

 

 

DOPAC
A 16
a:
E 14
a 12
5 10
3 8 * *
6 fi‘
8 4
0 2
0 1 I— ‘I I I I I
0 50 100 150 200 250 300
Apocynin (mg/kg)
B.
DA
250 1
g: 200-' 1 % fi1
3 150 4’
< 1001
D
50 1
0 I I I f I I
0 50 100 150 200 250 300
Apocynin (mg/kg)
C.
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a 0.08- *
o 0.06 - *
< *
0
0.02 -
0.00 I I I I I I

 

 

0 50 100 150 200 250 300
Apocynin (mg/kg)

Figure 4-3. Apocynin dose dependently decreases DOPAC concentrations and reduces
the DOPAC/DA ratio in the striatum. Mice were injected with vehicle, 30, 100, or 300
mg/kg apocynin i.p. and sacrificed 2.5 min later, n=4. The concentrations of DOPAC
(Panel A) and DA (Panel B), and the DOPAC/DA (Panel C) at specified dosages. Data
points represent means and bars one SEM. *Significantly reduced DOPAC
concentrations or the DOPAC/DA ratio compared to vehicle treated mice.

135

DA & DOPAL
80 l

a

5 DOPAL
E

3

S

0 DA

 

 

 

0 15 30 45 60
Reaction Time (min)

   

 

 

 

 

 

B.
DA
DPBS
21g] IApo1 uM
330 . DApo 10 uM
$25 ' BApO 100 UM
< 20 -
o 15 .
10 ‘
5 - ND.
0
PBS DA DA 81
MAO B
C.
DOPAL
UPBS
33,3: IIApo 1 uM
530 - IApo 10 uM
_I ..
< 33 J E3Apo 100 uM
d
o 15 -
D 10 -
5 ' ND. ND.
0
PBS DA DA 8:

MAO B

Figure 4-4. Apocynin (Apo) does not inhibit MAO B in vitro in a cell free system. Time
course of MAO B conversion of DA to DOPAL (Panel A). Lack of an effect on DA
depletion (Panel B) or the synthesis of DOPAL (Panel C) by apocynin in the presence of
MAO B and DA 1 h following reaction initiation, n=3. Columns represent means and
bars one SEM.

136

Apocynin as a Neuroprotective Agent in the Sub-Chronic MPT P Model of PD

The concentrations of DA and its metabolites DOPAC and HVA were measured
in the striatum of mice treated with saline or sub-chronic MPTP (10 mg/kg, s.c., once a
day for 5 days) treatment and given either vehicle or apocynin (100 mg/kg, i.p. every 12
h) to determine if apocynin prevented the loss of NSDA neuronal terminals. As shown in
Figures 4-5 & 4-6, DA, DOPAC, and HVA concentrations were similar in control mice
given either vehicle or apocynin. DA concentrations were significantly decreased in
MPTP treated mice and MPTP treated mice given apocynin. DOPAC and HVA
concentrations in the striatum were also significantly decreased with MPTP treatment in
both vehicle and apocynin treated mice. The DOPAC/DA and HVA/DA ratios in the
striatum were not altered with apocynin treatment alone, and were significantly increased
with MPTP treatment in vehicle and apocynin treated mice (Figure 4-5; Panel C & Figure
4-6; Panel B). These results reveal that apocynin did not prevent the loss of NSDA
neuronal terminals, or the compensatory activity of remaining NSDA neurons typically
seen with sub-Chronic MPTP treatment.

The extent of microglial activation was assessed in the striatum using content of
membrane bound p67Phox. The activation of NADPH oxidase involves the translocation
of the cytosolic subunit p67Phox to the cell membrane (Bianca et al., 1999; Groemping
and Rittinger, 2005). The content of membrane bound p67Phox can be used to determine
if there is an increase in microglial activation with MPTP treatment (Wu et al., 2002; Wu
et al., 2003). As shown in Figure 4—7, Membrane bound p67Phox protein in the striatum
was similar between vehicle and apocynin treated mice given saline vehicle. MPTP

treatment induced a significant increase in membrane bound p67Phox protein in vehicle

137

treated mice, consistent with activation of NADPH oxidase (Figure 4-7, Panel A). In the
presence of apocynin this increase was not present indicating that apocynin reduced I
activation of NADPH oxidase.

The neurochemical concentrations of DA, DOPAC, and HVA were also measured
in the nucleus accumbens to determine if sub-Chronic MPTP treatment and/or apocynin
altered MLDA neuronal activity. As shown in Table 4—1 , the concentrations of DA were
not different in the nucleus accumbens following MPTP or apocynin treatment
suggesting that MLDA neuronal integrity was maintained with MPTP treatment.
DOPAC concentrations in the nucleus accumbens were significantly decreased with
MPTP treatment in vehicle treated mice, but not altered with MPTP treatment in the
presence of apocynin. The HVA concentrations in the nucleus accumbens were not
changed in the presence of MPTP or apocynin. The DOPAC/DA and HVA/DA ratios
were similar in MPTP and/or apocynin treatment compared to saline and/or vehicle
treated mice. These results suggest that sub-Chronic MPTP treatment does not alter
MLDA neuronal integrity or activity.

T IDA neuronal integrity and activity were also assessed in the presence of sub-
Chronic MPTP and/or apocynin treatment. DA concentrations were similar in the median
eminence between saline and sub-chronic MPTP treated mice within both vehicle and
apocynin groups of mice (Table 4-2). The concentrations of DOPAC and HVA in the
median eminence were also not altered with MPTP and/or apocynin treatment. The
activity of TIDA neurons as determined by the DOPAC/DA and HVA/DA ratios were

similar between saline treated mice given either vehicle or apocynin. These findings

138

indicate that sub-chronic MPTP or apocynin treatment do not alter TIDA neuronal

integrity or activity.

Lack of an Effect on Serotoninergic Integrity and Activity with Sub-Chronic MPT P or
Apocynin Treatment

The concentrations of 5HT were measured in the nucleus accumbens and median
eminence to determine if sub-chronic MPTP or apocynin treatment altered serotoninergic
neuronal integrity. The concentrations of 5HT were similar in mice treated with saline or
MPTP given vehicle or apocynin in both the nucleus accumbens and median eminence.
Apocynin treatment also did not affect 5HT concentrations in either axonal terminal
region. The SHLAA concentrations were also determined in both terminal regions and
were not different with either MPTP or apocynin administration. The ratio of
SHIAA/SHT in the nucleus accumbens and median eminence were also similar in the
presence or absence of MPTP and apocynin within each axonal terminal region (Table 4-
1 & 4-2). This data indicates that serotoninergic neuronal integrity and activity are

maintained in the presence of either sub-Chronic MPTP or apocynin treatment.

139

DA

300 - DSaiine
3250 . IMPTP
3200 .
C
< 150 -
D 100 -

 

 

 

   

 

Vehicle Apocynin

DOPAC

CISaiine
E 20 . IMPTP

 

 

 

 

 

 

Vehicle Apocynin

DOPAC] DA

0.20 - DSaline
I MPTP

< .

 

   

 

 

 

Vehicle Apocynin

Figure 4-5. Apocynin does not alter MPTP induced DA depletion in the striatum. Mice
were treated with saline or a sub-Chronic MPTP treatment paradigm (10 mg/kg once a
day for 5 days) and sacrificed 3 days later. Mice were treated with either vehicle or
apocynin (100 mg/kg, i.p.) twice a day prior to and following MPTP treatment.
Concentrations of DA (Panel A), DOPAC (Panel B), and the DOPAC/DA ratio (Panel C)
in the striatum. Columns represent means and bars one SEM. *Significant difference
between saline and MPTP treated mice.

140

HVA

A DSaline
g 20 . IMPTP

 

2’15- *

 

 

 

   

 

 

Vehicle Apocynin

HVA! DA

0.25 ‘
* DSaline
0.20 1 * IMPTP

<

D 0.15 ‘

E 0.10 '
0.05 -1
0.00

 

 

 

 

 

 

 

   

Vehicle Apocynin

Figure 4-6. Apocynin does not alter MPTP induced HV A depletion in the striatum. Mice
were treated with saline or a sub-Chronic MPTP treatment paradigm (10 mg/kg once a
day for 5 days) and sacrificed 3 days later. Mice were treated with either vehicle or
apocynin (100 mg/kg, i.p.) twice a day prior to and following MPTP treatment.
Concentrations of HVA (Panel A) and the HVA/DA ratio (Panel B) in the striatum.
Columns represent means and bars one SEM. *Significant difference between saline and
MPTP treated mice.

141

100 '
80 ‘
60 a
40 ‘
20 1

RDU

 

 

p67Phox

*

 

 

p67Phox 67 kDa ——>

Vehicle

 

Apocynin

 

D Saline
I MPTP

Vehicle Apocynin
Saline MPTP Saline up'rp

SNAP-25 27 kDa — c- - - «_-

Figure 4-7. Membrane bound p67Phox protein in the striatum. Mice were treated with
saline or a sub-chronic MPTP treatment paradigm (10 mg/kg once a day for 5 days) and
sacrificed 3 days later. Mice were treated with either vehicle or apocynin (100 mg/kg,
i.p.) twice a day prior to and following MPTP treatment. p67Phox protein normalized to
the membrane protein SNAP-25 and multiplied by a factor of 1000 and expressed as
relative density units (RDU) (Panel A). Pictorial representation of p67Phox and SNAP-
25 protein bands from each respective treatment group (Panel B). Columns represent
means and bars one SEM. *Significant difference between saline and MPTP treated

mice.

142

Table 4-1. Neurochemical Concentrations in the Nucleus Accumbens Following Sub-
Chronic MPTP and Apocynin Treatment

 

Nucleus Saline/Vehicle MPTP/Vehicle Saline/Apocynin MPTP/Apocynin
Accumbens

 

DA 117.5 :1: 6.0 89.9 :1: 10.4 104.1 3: 11.5 92.0 :t 11.8
DOPAC 13.5:t1.2 8.7i0.8* 10.8:1: 1.1 9.1 :h 1.0

DOPAC/DA 0.12 i 0.01 0.10 :i: 0.01 0.11 :l: 0.01 0.11:t 0.2
HVA 12.0 i 0.4 10.4 :1: 0.6 10.9 i 0.6 10.0 :i: 0.7
HVA/DA 0.11:1:0.01 0.13:1:0.02 0.11:1:0.01 0.13:1:0.04
5HT 10.3 3: 1.6 10.8 +1.0 10.3 d: 1.4 10.7 +1.4
SHIAA 3.5 :t 0.5 4.6 i 0.5 3.4 :l: 0.4 3.9 :i: 0.6

SHIAA/SHT 0.35 :l: 0.02 0.44 :i: 0.04 0.35 :l: 0.02 0.38 :i: 0.04

 

Table 4-1. Mice were treated with saline or a sub-chronic MPTP treatment paradigm (10
mg/kg once a day for 5 days) and sacrificed 3 days later. Mice were treated with either
vehicle or apocynin (100 mg/kg, i.p.) twice a day prior to and following MPTP treatment.
The concentrations of DA, DOPAC, HVA, 5HT, SHIAA, and the DOPAC/DA,
HVA/DA, and SHIAA/SHT ratios were determined in the nucleus accumbens. Values are
the mean i the SEM. *Significant difference between saline and MPTP treated mice
within either vehicle or apocynin treatment groups.

143

Table 4-2. Neurochemical Concentrations in the Median Eminence Following Sub-
Chronic MPTP and Apocynin Treatment

 

 

Median Saline/Vehicle MPTP/Vehicle Saline/Apocynin MPTP/Apocynin
Eminence

DA 112.2:t 11.6 l46.9d:5.9 119.23: 8.0 116.3:t 16.3
DOPAC 7.1 i 0.5 6.5 i 0.3 7.7 :t 0.4 6.6 :h 0.7
DOPAC/DA 0.07 :t 0.01 0.05 :1: 0.01 0.07 :1: 0.01 0.06 a: 0.01
HVA 11.7:t0.8 14.8:1: 1.3 11.7:I:0.8 12.9i 1.1
HVA/DA 0.11:1: 0.01 0.10 :1: 0.01 0.10 :h 0.01 0.13 :l: 0.03
5HT 13.3i1.3 12.6i1.4 14.5i2.8 13.4:t2.0
SHIAA 14.0+1.4 15.7i0.5 13.6:t1.3 13.9i1.2
SHIAA/5HT 1.08+0.10 1.31 +0.08 1.05i0.11 1.10:1:0.09

 

Table 4-2. Mice were treated with saline or a sub-Chronic MPTP treatment paradigm (10
mg/kg once a day for 5 days) and sacrificed 3 days later. Mice were treated with either
vehicle or apocynin (100 mg/kg, i.p.) twice a day prior to and following MPTP treatment.
The concentrations of DA, DOPAC, HVA, 5HT, SHIAA, and the DOPAC/DA,
HVA/DA, and SHIAA/5HT ratios were determined in the median eminence. Values are
the mean i the SEM.

144

Apocynin as a Neuroprotective Agent in the Repeated Acute MPT P Model

The rapid elimination of measurable apocynin from the brain and the finding that
apocynin failed to attenuate or prevent the loss of NSDA axon terminals suggested that
apocynin administration may need to be delivered continuous over time rather than twice
daily. Although p67Phox membrane was significantly increased following sub-chronic
MPTP treatment, lack of a neuroprotective effect may be due to limited microglial
activation. Therefore, if microglial activation is limited, the MPTP induced loss of
axonal terminals in the striatum may be due exclusively to neurotoxic effects of MPTP
without the additive effects of microglial mediated cell death. The use of the repeated
acute MPTP model was employed to induce a more severe neurotoxic insult over a
shorter period of time and a greater extent of microglial activation. Due to its short half-
life, apocynin (250 mg/kg per day) was administered continuously via osmotic mini-
pump to maintain constant levels of apocynin in the brain.

The repeated acute MPTP model consists of injections of either saline vehicle or
MPTP ( 16 mg/kg, i.p.) every 2 h over a 6 h period for a total of 4 injections of MPTP,
and mice were killed 2 days later when microglial activation is maximal (Wu et al.,
2003). The activity of NADPH oxidase was measured in the striatum and ventral
midbrain of mice following saline or MPTP administration using brain homogenates
from each region. Repeated acute MPTP treatment induced a significant increase in
NADPH oxidase activity in the striatum (Figure 4-8; Panel A) that was more pronounced
than in the ventral midbrain (Figure 4-8; Panel B). An increase in NADPH oxidase

activity indicates that repeated acute MPTP induces a significant activation of microglia.

145

Striatum

CID-#010)

N

 

NADPH Oxrdase
Activity (RLU/minlug)

0...;

Saline MPTP

Ventral Midbrain

NADPH Oxrdase
Activity (RLU/minlug)
O —l N (A) A 01 O)

Saline MPTP

Figure 4-8. NADPH oxidase activation following repeated acute MPTP treatment. Mice
were treated with either saline or a repeated acute MPTP paradigm (4 injections of 16
mg/kg MPTP given i.p. every 2 h over a 6 h period). NADPH oxidase activity in the
striatum (Panel A) and ventral midbrain (Panel 13). Columns represent means and bars
one SEM. *Significant increase with repeated acute MPTP treatment versus saline.

146

In a subsequent experiment, mice were treated with apocynin (250 mg/kg per day)
beginning 2 days prior to repeated acute MPTP treatment and continued for an additional
2 days until the time of decapitation. The concentrations of DA were measured in the
striatum of saline and MPTP treated mice given either vehicle solution or apocynin via
osmotic mini-pump. DA concentrations were significantly decreased following MPTP
treatment to a similar extent in both vehicle and apocynin treated mice (Figure 4-9; Panel
A). MPTP induced a significant decrease in the content of TH in the striatum, which was
not attenuated with apocynin administration (Figure 4-10). These results indicate that
apocynin does not prevent a loss of NSDA axonal terminals seen with repeated acute
MPTP treatment.

DOPAC concentrations and the DOPAC/DA ratio (Figure 4-9; Panels B & C)
were examined in the striatum to determine if apocynin altered MPTP induced increases
in NSDA neuronal activity. DOPAC concentrations were significantly decreased to a
similar extent following repeated acute MPTP treatment in both vehicle and apocynin
treated mice. MPTP induced a significant increase the DOPAC/DA ratio in both vehicle
and apocynin treated mice (Figure 4—9; Panel C). These results indicate that apocynin
does not attenuate the compensatory activity of NSDA neurons seen with repeated acute
MPTP treatment.

NADPH oxidase activation was evaluated in the striatum following repeated acute
MPTP treatment by measuring membrane bound p67Phox and gp91Phox proteins.
MPTP treatment significantly induced an increase p67Phox and gp91Phox proteins in the
striatum of vehicle treated mice (Figure 4-11). A similar increase was seen in both

MPTP and apocynin treated mice (Figure 4-11). Thus, apocynin failed to block MPTP

147

induced translocation of p67Phox to the membrane, or increase in gp91Phox at the

membrane of microglial cells.

MLDA Neuronal Integrity and Activity with Repeated Acute MPT P and Apocynin
Treatment

DA and DOPAC concentrations were also measured in the nucleus accumbens
following repeated acute MPTP treatment (Table 4-3). MPTP induced a significant
decrease in DA and DOPAC concentrations, which was not different in MPTP/apocynin
treated mice. The DOPAC/DA ratio was not altered with MPTP administration in
vehicle treated mice, but was significantly increased following MPTP treatment in mice

given apocynin.

Serotoninergic Neuronal Integrity and Activity with Repeated Acute MPT P and Apocynin
Treatment

5HT and SHIAA concentrations in the nucleus accumbens were also measured to
determine if repeated acute MPTP and/or apocynin treatment affected serotoninergic
neuronal integrity or activity (Table 4-3). The concentrations of 5HT were significantly
decreased with MPTP treatment in mice given either vehicle or apocynin suggesting a
loss of serotoninergic axonal terminals in the nucleus accumbens. The SHIAA
concentrations were similar in the presence or absence of MPTP and/or apocynin
treatment. However, serotoninergic neuronal activity was increased in the nucleus

accumbens of MPTP/apocynin treated mice.

148

DA

250 -
DSaIine
a 200 - IMPTP

£1504
2’

:100'
0 50- * *

 

 

 

 

 

Vehicle Apocynin

DOPAC

20 -
El Saline

I MPTP

A
01
n

DOPAC(nglmg)
or 8

 

 

 

   

 

O

 

Vehicle Apocynin

DOPAC] DA

0'30 ' DSaiine
< 0.25 - * * IMPTP
Q 0.20 «

2 0.15 -
8 0.10 ~
a 0.05 «

0.00

 

   

 

 

 

 

Vehicle Apocynin

Figure 4-9. Apocynin does not alter repeated acute MPTP induced DA depletion in the
striatum. Mice were treated with either saline or a repeated acute MPTP treatment
paradigm (4 injections of 16 mg/kg MPTP given every 2 h over a 6 h period) and given
vehicle or apocynin 250 mg/kg per day via osmotic mini-pump. Concentrations of DA
(Panel A), DOPAC (Panel B), and the DOPAC/DA ratio (Panel C) in the striatum 2 days
following saline/MPTP treatment. Columns represent means and bars one SEM.
*Significant difference between saline and MPTP treated mice.

149

   

 

 

   

 

TH
30 -
25 J CJSaiine
3 201 IMPTP
o r
a: 15
10 -
5 .. * *
0
Vehicle Apocynin
B.
Vehicle Apocynin
Saline MPTP Saline MPTP
Beta "1 TUbunn 50 kDa ___-I. ~ -. -—- o——-. cg—fl

Figure 4-10. Loss of TH protein content in the striatum with repeated acute MPTP
treatment is not attenuated with apocynin. Mice were treated with either saline or a
repeated acute MPTP paradigm (4 injections of 16 mg/kg MPTP given every 2 h over a 6
h period) and given either vehicle or apocynin 250 mg/kg per day via osmotic mini-
pump. TH protein content was normalized to B—III tubulin protein content (Panel A).
Pictorial representation of TH and B-III tubulin bands for each treatment group (Panel B).
Columns represent means and bars one SEM. *Significant difference between saline and
MPTP treated mice. ‘

150

 

p67Phox

 

 

 

20-
315‘
0
K10.

 

 

U Saline

I MPTP
*

 

 

 

Vehicle Apocynin
gp91 Phox
El Saline
* * IMPTP

 

 

p67Phox 67 kDa ——->

gp91Phox 91 kDa —>

 

 

 

Vehicle

Apocynin

Vehicle Apocynin
Saline MPTP Saline MPTP

«ah-kw h m . fl

SNAP-25 27 kDa —-—> - - ‘-

Figure 4-1 1. Apocynin does not reduce p67Phox translocation to the membrane or an
increase in gp91Phox subunit membrane expression in the striatum observed with
repeated acute MPTP treatment. Mice were treated with either saline or a repeated acute
MPTP paradigm (4 injections of 16 mg/kg MPTP given every 2 h over a 6 h period) and
given vehicle or apocynin 250 mg/kg per day via osmotic mini-pump. p67Phox and
gp91Phox subunit expression were normalized to the membrane protein SNAP-25 and
multiplied by a factor of 1000. p67ph0x (Panel A) and gp91Phox (Panel B) protein
expression in the striatum membrane fraction. Pictorial representation of p67Phox,
gp91Phox, and SNAP-25 bands in treatment group (Panel C). Columns represent means
and bars one SEM. *Significant difference between saline and MPTP treated mice.

Table 4—3. Neurochemical Concentrations in the Nucleus Accumbens Following
Repeated Acute MPTP and Apocynin Treatment

 

Nucleus Saline/Vehicle MPTP/Vehicle Saline/Apocynin MPTP/Apocynin
Accumbens

 

DA 130.0:1: 10.5 81.1i10.4* 111.9:i:5.7 73.0:i:7.8*
DOPAC 15.8 +1.4 9.3 :1: 0.6 * 13.2 +1.3 10.1 :t 0.9 *
DOPAC/DA 0.12 :l: 0.01 0.13 :t 0.01 0.12 :h 0.01 0.15 :1: 0.02 *
5HT “2+ 1.1 7.9+0.9 * 12.3 +0.9 8.03:0.9 *
SHIAA 4.7 i 0.2 3.6 :l: 0.3 4.9 :l: 0.5 4.6 :l: 0.5
SHIAA/5HT 0.44 i 0.02 0.50 :i: 0.05 0.41 :l: 0.05 0.60 :1: 0.08 *

 

Table 4-3. Mice were treated with saline or a repeated acute MPTP paradigm (4
injections of 16 mg/kg MPTP given i.p. every 2 h over a 6 h period) and given vehicle or
apocynin 250 mg/kg per day via osmotic mini-pinup. The concentrations of DA,
DOPAC, 5HT, SHIAA, and the DOPAC/DA and SHIAA/5HT ratios were determined in
the nucleus accumbens. Values are the mean i the SEM. *Significant difference between
saline and MPTP treated mice within either vehicle or apocynin treatment groups.

152

D. Discussion

The use of apocynin as a neuroprotective agent for NSDA neurons in the MPTP
model of PD was examined due to its pharmacological property of inhibiting NADPH
oxidase. NADPH oxidase is a key enzyme responsible for the production of superoxide
by microglial cells (Pawate etal., 2004). Activation of NADPH oxidase is involved in
the microglial mediated death of NSDA neurons due to its role in the production of
reactive oxygen and nitrogen species and pro-inflammatory cytokines, and induction of
microglial proliferation and migration (Pawate et al., 2004; J ekabsone et al., 2006;
Mander et al., 2006). Indeed, mice lacking a functional gp91Phox subunit (the primary
membrane bound subunit) are partially resistant to both MPTP and LPS induced loss of
NSDA neurons (Wu et al., 2003; Qin et al., 2004). Apocynin is considered an ideal
candidate for neuroprotection of NSDA neurons because its lipophilic properties allows it
to cross the blood brain barrier readily and it has a high lethal dose (LD50) when 50% of
animals die due to toxicity (Van den Worm et al., 2001). Apocynin also reduces
oxidative stress, microglial activation, and loss of neurons seen in in vitro and/or in vivo
models of stroke, amyotrophic lateral sclerosis, Alzheimer’s disease, and PD, which
strongly supported its potential as a neuroprotective agent (Gao et al., 2003; J ekabsone et
al., 2006; Rey et al., 2006; Wang et al., 2006; Rodriguez-Pallares et al., 2007; Tang et al.,
2007; Harraz et al., 2008; Tang et al., 2008).

In the present study, the pharmacokinetics of apocynin in the brain were
examined to determine if apocynin crosses the blood brain barrier and to establish the
length of time it was present in the brain following systemic administration. The

measurement of apocynin in the brain using HPLC-EC in these studies was a novel

153

method in the laboratory. The results indicated that apocynin readily crosses the blood
brain barrier within 2.5 nrin following drug administration. Surprisingly, apocynin was
hardly detectable after 20 min in the brain following administration of a 100 mg/kg dose
to mice. This result was unexpected since apocynin is reported to reduce superoxide
production and neuronal death within the brain for a much longer period at even lower
doses, but unfortunately, these studies did not measure the concentrations of apocynin
within the brain (Rey et al., 2006; Wang et al., 2006). Recently a metabolite of apocynin
known as diapocynin has been implicated as a more potent inhibitor of NADPH oxidase
than apocynin in vitro (Ximenes et al., 2007). Rapid metabolism of apocynin in the brain
may therefore explain why apocynin can only be measured in the brain for such a short
period of time in this study. The ability of apocynin metabolites to inhibit NADPH
oxidase would explain the potential reduction of superoxide production in the brain long
after it was no longer measurable.

The potential time and dose dependent effects of apocynin on NSDA neuronal
activity were also examined by measuring the concentrations of DA and DOPAC within
the striatum. DA concentrations were not altered with apocynin treatment at any time
point. DOPAC concentrations and the DOPAC/DA ratio were significantly decreased in
a dose dependent manner by 2.5 min following apocynin administration. These findings
could be attributed to one potential cause, that apocynin may inhibit the metabolism of
DA to DOPAC by blocking MAO B activity which has not been described in the
literature. This hypothesis of MAO B inhibition was tested using an in vitro cell free
assay system measuring the catabolism of DA to DOPAL in the presence of apocynin.

The results from this study reveal that apocynin does not inhibit MAO B.

154

The implication of MAO B inhibition in the presence of MPTP treatment is that
apocynin could inhibit the metabolism of MPTP to its active metabolite MPP+. This
possibility could indicate a neuroprotective property of apocynin that would be attributed
to NADPH oxidase inhibition, but rather could also be due to a reduced neurotoxic effect
of MPTP.

Although the half-life of apocynin is relatively short in the brain, it was decided
that apocynin would be given 2 h prior to MPTP administration, since other studies
which revealed a neuroprotective effect of apocynin administered the drug at least 30 min
prior to neurological insult. Apocynin (100 mg/kg, i.p.) was injected twice a day 12 h
apart beginning 2 days prior to the initiation of sub-chronic MPTP administration and
continued up to the morning the mice were killed. MPTP treatment induced a significant
and similar loss of DA in the striatum with either vehicle or apocynin treatment
indicating that apocynin failed to attenuate the loss of axon terminals. Apocynin did not
alter the increased activity of NSDA neurons seen with MPTP treatment, which is due to
the compensation of the remaining unlesioned neurons for the loss of axon terminals
(Sundstrom et al., 1987; Drolet et al., 2004). These findings contradict others that
showed apocynin to be neuroprotective in a rat model of PD using the neurotoxin 6-
OHDA (Rey et al., 2006).

Lack of a neuroprotective effect of apocynin in the sub-chronic MPTP model
could be due to a limited amount of microglial induced loss of NSDA axonal terminals in
this MPTP model. Another possibility is that apocynin (or potentially one of its active

metabolites) may not be available for sufficient time to cause a significant reduction in

NADPH oxidase activity and subsequent microglial mediated cell death. However, in the

155

present study apocynin administration blunted MPTP-induced NADPH oxidase
activation of membrane bound p67Phox in the striatum suggesting that this was not the
case.

The increase in p67Phox at the membrane seen with MPTP treatment though
significant may still be below the threshold to observe any neuroprotective effects of
apocynin. Another MPTP model, the repeated acute MPTP model was therefore
employed as it induces significant microglial activation (demonstrated by increased
p67Phox translocation) within 2 days following a series of 4 injections of MPTP (16
mg/kg) over a 6 h time period (Wu et al., 2002; Wu et al., 2003). Continuous delivery of
apocynin using an osmotic mini-pump was used with the repeated acute MPTP model in
an effort to maintain apocynin concentrations in the brain. The idea for this method of
MPTP and apocynin administration was that in the presence of a more significant
lesioning of NSDA neuronal terminals and microglial reaction, apocynin would be more
likely to exhibit a neuroprotective effect.

Indeed, repeated acute MPTP treatment increases the amount of NADPH oxidase
activity as determined by an in vitro NADPH oxidase activity assay using brain tissue
from saline and MPTP treated mice and by an increase in membrane bound p67Phox
agreeing with others (Wu et al., 2002; Wu et al., 2003). The membrane bound subunit
gp91Phox (which is critical for NADPH oxidase production of superoxide) was also
significantly increased with MPTP treatment as others have shown in an animal model of
PD and human PD brains (Wu et al., 2003), which suggests either an upregulation of the

protein or an increase in microglial number.

156

The repeated acute MPTP model also induces a significant decrease in DA
concentrations and TH protein in the striatum of vehicle treated mice demonstrating a
loss of NSDA axonal terminals. However, even with constant infusion (250 mg/kg per
day), apocycnin failed to attenuate the loss of NSDA neuronal terminals as indicated by a
similar loss in DA and TH protein with MPTP treatment compared to MPTP/vehicle
treated mice. Similarly, membrane p67Phox was increased with repeated acute MPTP
treatment, but delivery of apocynin using the osmotic mini-pump did not reduce NADPH
oxidase activation with MPTP treatment.

Although MPTP clearly induced activation of NADPH oxidase it is possible that
with infusion of apocynin the concentrations present in the brain at any one time were too
low to inhibit the enzyme, but others have shown that at dosages much lower (apocynin 3
mg/kg per day) osmotic mini-pump delivery of apocynin inhibited NADPH oxidase
mediated effects (Zhan et al., 2005). Another possibility in regard to the dosage is that
there may be a narrow therapeutic window of apocynin, since this compound is reported
to be neuroprotective in a stroke model at 2.5 mg/kg (but not at 3.75 mg/kg) when
administered intravenously 30 min before the stroke was induced (Tang et al., 2008).

The integrity and activity of MLDA and TIDA neurons was also examined with
MPTP and apocynin treatment. The findings that apocynin itself did not alter MLDA or
TIDA neuronal terminal integrity or neuronal activity has not previously been noted in
the literature. These results are not surprising in that apocynin injection did not alter the
NSDA neurons activity following acute injection or with mini-pump administration.

Sub-chronic MPTP treatment did induce a slight decrease in DOPAC

Concentrations in the nucleus accumbens, but otherwise failed to alter the integrity or

157

activity of NSDA or MLDA neurons demonstrating that these neurons are resistant to
MPTP treatment with this dosing paradigm. In contrast, repeated acute MPTP treatment
induced a significant loss of MLDA axon terminals and cause increased compensatory
neuronal activity in the presence of both MPTP and apocynin. Repeated acute MPTP
treatment clearly leads to a more severe lesioning of dopaminergic neurons than the sub-
chronic MPTP paradigm.

The integrity and activities of serotoninergic neurons terminating in the nucleus
accumbens and median eminence were evaluated in the presence of apocynin, since there
has not been any reports as to any potential effects or lack of apocynin on serotoninergic
neurons in the central nervous system. Similar to dopaminergic neurons, apocynin did
not alter serotoninergic neurons with either an acute apocynin injection or delivery by
osmotic mini-pump.

MPTP treatment typically either does not effect serotoninergic neurons or only to
a minimal extent in high dosing paradigms of MPTP in mice (Hallman et al., 1985; Date
etal., 1990; Rousselet etal., 2003). Indeed, sub—Chronic MPTP treatment did not alter
serotoninergic terminals or neuronal activity in either the nucleus accumbens or median
eminence. In agreement with the more severe MPTP paradigms inducing a loss of
serotoninergic neuronal terminals (Rousselet et al., 2003), repeated acute MPTP
treatment induced a significant loss in 5HT concentrations within the nucleus accumbens.
The SHIAA concentrations were also decreased with MPTP treatment with this
paradigm, and with MPTP/apocynin treatment the SHIAA/5HT ratio was increased

indicating an increase in the activity of remaining serotoninergic neuronal terminals.

158

These studies are in agreement with others showing that NADPH oxidase
activation increases with MPTP treatment due to indirect activation of microglia by the
direct neurotoxin induced loss of NSDA neurons (Gao et al., 2003). The production of
superoxide and consequent formation of other reactive oxygen species and induction of
iNOS and pro-inflammatory cytokines is incredibly detrimental to NSDA neurons (Wu et
al., 2002; Wu et al., 2003). The use of the NADPH oxidase inhibitor apocynin seemed
therefore to be an ideal method to reduce microglial NADPH oxidase mediated death of
NSDA neurons. This theory was supported by others who demonstrated that apocynin
was neuroprotective in models of other diseases associated with microglial NADPH
oxidase induced death of neurons (Rey et al., 2006; Wang et al., 2006). However,
complications of the complex pharmacokinetics of apocynin and little knowledge about
its proposed active metabolites made it Challenging to Choose the appropriate dosage,
frequency, and method of administration of the drug. The equivalent loss of NSDA
axonal terminals seen with apocynin treatment in the face of MPTP induced microglial
activation, therefore indicates that apocynin was not neuroprotective in either the sub-

Chronic or repeated acute MPTP models of PD.

159

Chapter 5: The Characterization of the Neurochemical Pr0perties and
N euromodulation of Dopaminergic Neurons in Wildtype and Cannabinoid 1 and 2

Receptor Knockout Mice

A. Introduction

Endocannabinoid (ECB) regulation of DA neuronal populations within the central
nervous system is complex, and the effects of ECB on dopaminergic function vary by
population. ECB regulate NSDA, MLDA, and TIDA neurons either directly or indirectly
through afferent upstream neuronal systems.

ECB are involved in the regulation of NSDA neurons, but these neurons do not
express CBl receptors (Herkenham et al., 1991a). The presence of CB] receptors
throughout the basal ganglia and ECB action at both glutamatergic and GABAergic
synapses within the basal ganglia leads to the complexity of ECB regulation of NSDA
neurons (Figure 5-1). CB can facilitate NSDA neuronal activity by increasing the firing
rate of the neurons and release of DA using a CB1 receptor mediated mechanism within
the basal ganglia (French et al., 1997; Melis et al., 2000; Price et al., 2007; Morera-
Herreras et al., 2008). Cannabinoids (CB) in contrast can also inhibit synthesis of DA
and decrease its release from NSDA neurons (Cadogan et al., 1997; Moranta et al., 2004).
The differential roles CB have in NSDA neuronal regulation are likely due to actions at

different populations of CB1 receptors within the basal ganglia.

160

Cannabinoid Receptor
Expression in the Basal Ganglia

   
  

    

Direct ‘
Pathway

  

Figure 5-1. CB1 receptor expression within the basal ganglia. CB1 receptors are located
on both glutamatergic (A) and GABAergic (-L) terminals within the basal ganglia. CBl
receptors are on glutamatergic cortical neurons terminating in _the striatum. Medium
spiny GABAergic neurons projecting to the globus pallidus interna (GPi), substantia
nigra pars reticulata (SNpr), and globus pallidus extema (GPe) have axonal terminal CB1
receptors. Glutarnatergic neurons originating in the subthalamic nucleus (STN) and
terminating in the GPi and SNpr also have terminal CB1 receptors. ECB action at
different locations within the basal ganglia can alter the activity of the direct (excitatory)
pathway or the indirect (inhibitory) pathway on the thalamus, and also indirectly effect
NSDA neurons originating in the substantia nigra pars compacta (SNpc) and terminating
in the striatum.

161

MLDA neurons comprise the dopaminergic component portion of the reward
pathway and increased DA release contributes to the addictive properties of several drugs
of abuse including alcohol, nicotine, and cocaine. CB increase MLDA neuronal activity
and release of DA into the nucleus accumbens (French et al., 1997; Gessa et al., 1998;
Cheer et al., 2004; Melis et al., 2004b; Riegel and Lupica, 2004), which is likely due to
modulation of depolarization induced suppression of GABA and/or glutamate release
within the ventral tegrnental area. Suppression of GABA or glutamate release occurs
through release of ECB from MLDA neurons that feedback onto CBl receptors within
the ventral tegmental area resulting in increased activity of MLDA neurons (Melis et al.,
2004a; Riegel and Lupica, 2004). CB and ECB induction of DA release in the nucleus
accumbens is reversed in the presence of the CBI antagonist rimonabant indicating the
importance of ECB in MLDA neuronal function (Gessa et al., 1998; Melis et al., 2000;
Cheer et al., 2007).

TIDA neurons inhibit secretion of the hormone prolactin from the anterior
pituitary by releasing DA from the median eminence into the hypophyseal-portal system
that transports DA to the anterior pituitary (Sellix and Freeman, 2003). CB1 receptors
are present in the arcuate nucleus and median eminence implicating ECB in the
regulation of TIDA neurons (Wittrnann et al., 2007). The administration of the ECB
anandamide differentially regulates prolactin secretion depending on sex and estrogen
levels, indicating that ECB indeed actively regulate TIDA neurons. Anandamide
significantly increases the release of prolactin in ovariectomized female rats given
estrogen replacement that is accompanied by a reduction in TIDA neuronal activity.

Ovariectomized female rats without estrogen replacement do not have altered prolactin

162

levels, but do have reduced TIDA neuronal activity following anandamide treatment
(Scorticati et al., 2003). In contrast, male rats demonstrate a reduction in prolactin
release following anandamide treatment that corresponds to an increase in activity of
TIDA neurons (Scorticati et al., 2003). The sex and hormonal differential regulation of
TIDA neurons by ECB suggests that estrogen and other hormones or neurotransmitters
may play a role in the effects of ECB on TIDA neurons.

ECB can also regulate serotoninergic neurons within the central nervous system.
5HT neuronal cell bodies are located in the dorsal and median raphe nuclei of the
brainstem and project to regions throughout the brain including to the striatum and
nucleus accumbens (Moore et al., 197 8). CB1 receptors are localized on serotoninergic
neurons within the raphe nuclei, and CB decrease the synthesis of 5HT in the striatum
and reduce 5HT release in the nucleus accumbens and cortex via a CB1 receptor
dependent mechanism (Nakazi et al., 2000; Moranta et al., 2004; Sano et al., 2008). CB
can also increase 5HT neuronal activity and reduce depression which is typically
associated with lower 5HT levels in the brain (Bambico et al., 2007). The differential
findings concerning 5HT neuronal function indicates that (similar to dopaminergic
neurons) serotoninergic neurons may be regulated by more than one CB mechanism.

The role of CB receptors in the central nervous system has mainly been studied in
C81 KO mice because of the abundant expression of C31 receptors in the brain. CBl
KO mice have TH mRN A expression similar to WT mice within the substantia nigra and
ventral tegrnental area (the cell body regions of NSDA and MLDA neurons) and have
similar DA concentrations in the nucleus accumbens (Steiner et al., 1999; Hungund et al.,

2003). This finding suggests that CBl KO mice have normal NSDA and MLDA

163

neuronal integrity compared to WT mice. CB1 KO mice also have normal expression of
mRNA for the D2 receptor in the striatum suggesting that CBl KO mice may have intact
D2 receptor regulation of NSDA neurons (Gerald et al., 2006). Evaluation of the motor
activity of CB1 KO mice reveals that they have normal motor coordination, but are
hypoactive in an exploratory behavior test and experience increased catalepsy (Steiner et
al., 1999; Zimmer et al., 1999). Catalepsy (inhibition of movement) is typically a
consequence of CB treatment and thus contradicts findings in the CB1 KO mice that
would be expected to have increased locomotion.

CBZ KO mice dopaminergic neuronal integrity and activity have not previously
been studied to our knowledge. However, CB2 KO mice are more prone to induction of
inflammation within the brain in a rodent model of multiple sclerosis (Maresz et al.,
2007). Therefore since the integrity and function of dopaminergic and serotoninergic
neurons in CB1/CB2 KO mice is unknown, the Characterization of the integrity and

activity of these neuronal populations in CB1/CB2 KO mice is of interest.

164

Hypothesis #1: A lack of functional CB1 and CB2 receptors will alter the integrity of
NSDA, MLDA, TIDA, or serotoninergic neurons.
This hypothesis was tested by examining:

1) The concentrations of DA in the terminal regions of NSDA, MLDA, and TIDA
neurons (striatum, nucleus accumbens, and median eminence, respectively) as an
index of the integrity of dopaminergic neuron axon terminals.

2) The integrity of NSDA neuronal cell bodies and axon terminals by assessing the
number of TH immunoreactive cells within the substantia nigra and protein
content of TH in the ventral midbrain (cell bodies) and TH and DAT in the
striatum (neuronal terminals).

Hypothesis #2: The activity and/or regulation of dopaminergic and serotoninergic
neurons will be altered in mice lacking functional CB1 and CB2 receptors.
This hypothesis was tested by examining:

1) The activity of NSDA, MLDA, and TIDA neurons by measuring the DA
metabolites DOPAC and HVA and determining the ratio of DOPAC/DA and
HVA/DA.

2) Dopaminergic receptor regulation of NSDA, MLDA, and TIDA neurons by
assessing long loop and short loop feedback regulation in CB1/C82 KO mice
using the concentrations of DOPAC and the ratio of DOPAC/DA as an index of
DA neuronal activity.

3) The activity and axon terminal integrity of serotoninergic neurons terminating in
the striatum and nucleus accumbens was examined by determining the

concentrations of 5HT and its metabolite SHIAA in the specified terminal regions.

165

B. Materials and Methods

Male C57BL/6 referred to as Wildtype (WT) mice were obtained from (Jackson
Labs) and used as controls for male CB1/CB2 receptor KO mice bred by our
collaborators at Michigan State University. Mice used for all experiments were between
8-12 weeks of age and did not differ in average body weight (Figure 5-2). In
neurochemical studies mice were sacrificed by decapitation and brain tissue processed for
neurochemistry or Western blot analysis as previously described (Chapter 2, Section C,
Euthanasia and Brain Tissue Preparation). In immunohistochemical studies hindbrains
were dissected at the mid-coronal plane following decapitation, and drop fixed and stored
in 4% paraformaldehyde for 7 days and placed in 20% sucrose for 24 h before being
processed. TH cell counts in the substantia nigra using unbiased stereology were done as
previously described (Chapter 2, Section H, Stereology TH Cell Counts).

D2 autoreceptor regulation in WT and CB1/C32 KO mice was tested by treating
mice with y-butyrolactone (GBL) (750 mg/kg; ip) alone or in combination with
quinelorane (0.1 mg/kg; ip) l h prior to decapitation. CB1/CB2 K0 and WT mice were
also treated with the D2 receptor antagonist raclopride to determine if CB1/C32 KO mice
had normal post-synaptic D2 receptor regulatory mechanisms. Mice were injected with
either saline vehicle or raclopride (1 mg/kg/ip) l h prior to decapitation. Brains were
immediately frozen on dry ice following removal of the median eminence for
neurocherrrical analysis as previously described (Chapter 2, Section C, Preparation of

Brain Tissue).

166

WT and CB1/C32 KO Weights

30-
25-
204
151
10-

5-

0-

Weight (g)

 

 

WT CB1/C32 KO

Figure 5-2. WT and CB1/CB2 KO mouse body weight (g), n=8. Columns represent
means and bars one SEM.

167

C. Results
Comparison of NSDA neurons in WT and CBI/CB2 Receptor K0 Mice

Several indices of NSDA neurons were evaluated in both WT and CB 1/CB2
receptor KO mice to determine if lack of functional CB receptors alters the integrity and
function of NSDA neurons. Basal levels of DA and its metabolites DOPAC and HV A in
the striatum (Figures 5-3 and 5-4) were not different in CB1/CB2 receptor KO mice. The
basal activity of NSDA neurons as indicated by the ratio of DOPAC/DA was equivalent
in both WT and CB1/C32 KO mice (Figure 5-3; Panel C). The ratio of HVA/DA was
similar in WT and CB1/CB2 KO mice suggesting that a lack of CB receptors does not
alter glial metabolism of DOPAC to HVA via the enzyme COMT (Figure 5-4; Panel B).

Vesicular DA concentrations measured within the striatum is used as an indirect
index of axonal integrity and density of innervation of NSDA neurons (Drolet et al.,
2004). The lack of a difference in DA concentration within the striatum was
complementary to other axonal terminal markers of NSDA neurons, which included
striatum protein expression of TH and DAT that were similar in WT and CB1/C82 KO
mice (Figure 5-5). Phosphoryiation of serine 40 on TH protein is the key step in
activation of TH synthesis of DA to replenish neurotransmitter released by NSDA
neurons. No difference in phosphorylated serine 40 TH was observed by Western blot
(Figure 5-5).

NSDA neuronal cell body integrity was evaluated in CB1/CB2 KO mice as
determined by analysis of TH protein expression in the ventral midbrain by Western blot
and the numbers of TH-immunoreactive cells within the substantia nigra. The amount of

TH protein expression in the ventral midbrain and distribution of TH immunoreactive

168

cells within the substantia nigra was comparable in CB1/C32 KO and WT mice (Figure
5-6). A higher powered (20 X) view of NSDA neuronal cell bodies demonstrated normal
cell morphology in CB1/CB2 KO mice (Figure 5-6). Unbiased stereology cell counts of
TH-immunoreactive cells within the bilateral substantia nigra demonstrated similar TH
cell numbers (Figure 5-7; Panel A). Western blot analysis of ventral midbrain TH protein

content (Figure 5-7; Panel B) did not differ between WT and CB1/CB2 KO mice.

169

DA

   

CB1/C32 KO

DOPAC

   

CB1/632 KO

DOPAC/DA

0.5

g 0.4
g 0.3
o 0.2
0.1
0.0

   

WT CB1/C32 KO

Figure 5-3. Concentrations of DA (Panel A) and DOPAC (Panel B) and the DOPAC/DA
ratio (Panel C) in the striatum of male WT and CB1/C32 KO mice, n=9-10. Columns
represent means and bars one SEM.

170

HVA (nglmg)
a a 8

01

 

O

 

CB1/082 KO

HVA/DA
0.10
0.08 i
<
S 0.06
E 0.04
0.02

0.00
WT CB1/(332 KO

Figure 5-4. Concentrations of HVA (Panel A) and the HVA/DA ratio (Panel B) in the

striatum of WT and CB1/CB2 receptor KO mice, n=7-8. Columns represent means and
bars one SEM.

171

 

 

 

 

   

TH
20 -
DWT
15 ~ ICB1/CBZ KO
3 .
o 10 -
r:
5 ..
o -
TH Serine 40 TH
B.
DAT
50 -
40 5
3 30 -
n:
20 .
10 .
o -
CB1/C32 KO
CO
WT CB WT CB
K0 K0
TH 60 kDa —> hi- DAT 70 kDa —> an an

Beta Ill Tubulin 50 kDa —# ill-\- SNAP-25 25 kDa—D —- -—

Serine 40 TH 60 kDa —>
Beta Iii Tubulin 50 kDa —> — _-

Figure 5-5. Expression of TH, phosphorylated serine 40 TH, and DAT in the striatum of
WT and CB1/CB2 KO mice as determined by Western blot. TH and phosphorylated
serine 40 TH protein expression normalized to B-IIl tubulin times a factor of 100 in the
striatum (Panel A) and DAT protein expression normalized to SNAP-25 times a factor of
1000 (Panel B) and expressed as relative density units (RDU) of protein bands, n=8-11.
Columns represent means and bars one SEM. Pictorial representation of TH,
phosphorylated serine 40 TH, B—III tubulin, DAT, and SNAP-25 protein bands from the
striatum of WT and CBl/CB2 KO mice (Panel C).

172

CB1/C82 KO

 

Figure 5-6. TH immunoreactive neurons within the substantia nigra of WT (Panels A &
B) and CB1/C82 KO mice (Panels C & D). Photomicrographs depict TH staining (in
brown) of neurons within the substantia nigra of WT and CB1/CB2 KO mice. Black
outline surrounds the area of the substantia nigra that was delineated to count TH cells
within in all animals (Panel A). Arrows appearing in (Panels B & D) indicate NSDA
neuronal cell bodies immunopositive for TH within the substantia nigra. Bar pictured in
(Panel C) is 500 uM and bar pictured in (Panel D) is 100 uM.

173

TH Cell Counts

.h
C
O
O

(.0
O
O
O

2000
1000

TH Cell Number

   

CB1/C32 KO

35
3O
25
20
15
10

RDU

   

WT CB1/C32 KO

Figure 5-7. Bilateral TH cell body stereological counts in the substantia nigra and TH
protein expression in the ventral midbrain of WT and CB1/CB2 KO mice. The number
of TH cells within the substantia nigra was determined by counting cells using an
unbiased stereology method (Panel A), n=3-7. TH protein expression in the ventral
midbrain (Panel B) normalized to B-III tubulin expression multiplied by a factor of 100
and expressed as relative density units (RDU) of protein bands, n=9-10. Columns
represent means and bars one SEM.

174

Comparison of MLDA and TIDA neurons in WTand CBI/CBZ K0 Mice

The concentration of DA and one or more of its metabolites was measured in the
terminal regions (nucleus accumbens and median eminence) of MLDA and TIDA
neurons. The concentration of DA and its metabolites DOPAC and HVA did not differ
between WT and CB1/CB2 KO mice in the nucleus accumbens (Figures 5-8; Panels A
&B and 5—9; Panel A). The DOPAC/DA and HVA/DA ratios were similar in both WT
and CB1/CB2 KO mice (Figures 5-8; Panel C and 5-9; Panel B).

In the median eminence the concentrations of DA and DOPAC was similar
between WT and CB1/C32 KO mice (Figure 5-10; Panels A & B). The DOPAC/DA
ratio was also similar in WT and CB1/CB2 KO mice indicating normal TIDA neuronal

activity in CBl/CB2 KO mice (Figure 5-10; Panel C).

175

DA

250~
@200-
3,150-
5100-
<
0 501

 

   

 

WT CB1/082 KO

DOPAC

 

   

WT CB1ICBZ KO

DOPAC] DA

0.20-f
<
9 0.15 4
U
3‘, 0.10 ~
0
0 0.05 -
0.00 -

 

   

WT CB1/082 KO

Figure 5-8. Concentrations of DA (Panel A) and DOPAC (Panel B) and the DOPAC/DA
ratio (Panel C) in the nucleus accumbens of WT and CB1/C32 KO mice. Columns
represent means and bars one SEM.

176

HVA

_n _s
o 01
J I

HVA (nglmg)
OI

 

 

O
I

CB1/082 KO

 

HVA/DA
0.20 -

0.15 -
$0.10 ~
>
I 0.05 -

 

 

 

0.00 -
WT CB1/082 KO

Figure 5-9. Concentrations of HVA (Panel A) and the HVA/DA ratio (Panel B) in the
nucleus accumbens of WT and CB1/CB2 KO mice. Columns represent means and bars

one SEM.

177

DA

200 1
150 '
100 1

501

DA (nglmg)

 

   

WT CB1/632 KO

DOPAC

DOPAC (nglmg)
O.)

 

   

CB1/032 KO

DOPAC] DA

0.05 .
< 0.04 -
Q
o 0.03 .
5002
o .

‘3 0.01 .

0.00 -

 

   

WT CB1/(282 KO

Figure 5-10. Concentrations of DA (Panel A) and DOPAC (Panel B) and the
DOPAC/DA ratio (Panel C) in median eminence of WT and CB1/CB2 KO mice.
Columns represent means and bars one SEM.

178

DA Autoreceptor Regulation of Central DA Neurons in WT and CBI/CBZ K0 Mice

GBL treatment in the presence of quinelorane is commonly used as a model to
study DA autoreceptor feedback regulation of dopaminergic neurons (Walters and Roth,
1976; Foreman et al., 1989; Eaton et al., 1994). Pre-synaptic D2 auto-receptor function
was evaluated in CB 1/CB2 KO mice by treating mice with either saline/vehicle, GBL
(750 mg/kg ip)/vehicle, or GBL/quinelorane (0.1 mg/kg i.p.). Vehicle or quinelorane
were administered 1 min prior to saline/GBL injection. In the striatum DA and DOPAC
concentrations were significantly increased in WT and CB1/CB2 KO mice to an equal
extent in the presence of GBL alone. These increases were significantly attenuated in the
presence of quinelorane restoring DA and DOPAC concentrations almost to control
levels in both WT and CB1/C32 KO mice (Figure 5-11; Panels A & B). The
DOPAC/DA ratio in WT and CB 1/CB2 KO mice was not Changed in the presence of
GBL alone, but when quinelorane was given prior to GBL there was a similar reduction
in the ratio for both WT and CB1/CB2 KO mice due to restoration of DA autoreceptor
feedback inhibition (Figure 5-11; Panel C).

In vehicle treated controls the concentration of DOPAC in the nucleus accumbens
was significantly higher in CBl/CBZ KO mice (Figure 5-12; Panel B). DA
concentrations were not Changed with GBL/vehicle or GBL/quinelorane treatment
compared to saline/vehicle treated WT or CB1/C82 KO mice. DOPAC concentrations
following GBL/vehicle treatment were not Changed in WT mice, but were significantly
lower in CB1/CB2 KO mice due to higher DOPAC concentrations in saline/vehicle .
CB1/CB2 KO mice. Both WT and CBZ/CB2 KO mice treated with GBL/quinelorane

had significantly lower DOPAC concentrations within the nucleus accumbens compared

179

to either saline/vehicle or GBL/vehicle treated mice. The DOPAC/DA ratio within the
nucleus accumbens was significantly decreased in both WT and CB1/CB2 KO mice in
the presence of either GBL/vehicle or GBL/quinelorane. However, the decrease with
GBL/quinelorane treatment was more pronounced than with GBL alone (Figure 5 -12;
Panel C).

As shown in Figure 5-13 (Panels A & B), GBL treatment alone failed to alter the
concentrations of DA or DOPAC in the median eminence of WT or CBl/CB2 KO mice.
DA and DOPAC concentrations were higher in CB1/C32 KO mice compared to WT
mice. The DOPAC/DA ratio was not different among any of the treatment groups in WT

or CB1/CB2 KO mice (Figure 5-13; Panel C).

180

 

 

 

 

 

 

 

 

 

 

    

 

 

 

DA
600 i DWT
3400 . *
a *A A
5 300 -
< 200 -
D
100 -
0
Saline! GBL! GBU
Vehicle Vehicle Quinelorane
B.
DOPAC
80 1
‘5” (758 . * ICB1/CBZ KO
3 :3-
2 30 - *A*l\
3 20 -
Q 10 '1
0
Saline! GBL! GBL!
Vehicle Vehicle Quinelorane
C.
DOPAC/DA
0.25 - DWT
< 0.20 - ICBl/CBZ KO
D
a 0.15 -
<
3 0.10 '1 *A*A
‘3 0.05 - I
0.00
Saline! GBU GBU
Vehicle Vehicle Quinelorane

Figure 5-11. D2 autoreceptor regulation in the striatum of WT and CB1/C32 KO mice.
Concentrations of DA (Panel A), DOPAC (Panel B), and the DOPAC/DA (Panel C) in
WT and CB1/CB2 KO mice given saline/vehicle, GBL (750 mg/kg)/vehicle, or
GBL/quinelorane (0.1 mg/kg) 1 h prior to decapitation, n=7-8. Columns represent means
and bars one SEM. *Saline/vehicle treated mice are significantly different from
GBL/vehicle or GBL/quinelorane treated mice. “GBL/quinelorane mice are significantly

different from GBL/vehicle treated mice p 5 0.05.

181

 

 

 

 

 

 

 

   

 

 

 

 

    

    
 

DA
250 - DWT
3200 . ICB1/CBZ KO
€150 -
5
g 100 1
50 1
0
Saline! GBL! GBU
Vehicle Vehicle Quinelorane
B.
DOPAC
DWT
ICBl/CBZ KO
*
*A
Mi.
Saline! GBU GBU
Vehicle Vehicle Quinelorane
C.
DOPAC/DA
0.4 - DWT
< 0.3 - ICBl/CB2 KO
9
0 0.2 -
3‘. *A *A
O 0.1 -
D

 

Saline! GBL! GBU
Vehicle Vehicle Quinelorane

Figure 5-12. D2 autoreceptor regulation in the nucleus accumbens of WT and CBl/CB2
KO mice. Concentrations of DA (Panel A), DOPAC (Panel B), and the DOPAC/DA
ratio (Panel C) in WT and CBl/CB2 KO mice given saline/vehicle, GBL (750
mg/kg)/vehicle, or GBL/quinelorane (0.1 mg/kg) 1 h prior to decapitation, n=6-8.
Columns represent means and bars one SEM. *Significant difference between
saline/vehicle and treatment groups within either WT or CB1/CB2 KO. A Significant
difference between GBL/vehicle and GBL/quinelorane groups for either WT or CB1/C32
KO mice. *Significant difference between WT and CB1/CB2 KO mice within a
treatment group p 5 0.05.

182

 

DA
400 1 ClWT
350 ‘ ICB1/CBZ KO
“300 - *
E 250 ‘
B200 -
2' 150 -
100 '1
D 50 -
o -
Saline! GBIJ GBL]

Vehicle Vehicle Quinelorane

 

 

 

 

 

 

DOPAC
315 ’ * uwr
E ICB1/CBZ KO
2 10 -
2
a 5 ‘
O
D
0
Saline! GBL! GBL!
Vehicle Vehicle Quinelorane
DOPAC/DA
0.08 - DWT
< 0 06 _ ICB1/CBZ KO
8
< 0.04 -
8
g 0.02 - I I l
0.00 -
Saline! GBU GBL!
Vehicle Vehicle Quinelorane

Figure 5-13. D2 autoreceptor regulation in the median eminence of WT and CB1/CB2
KO mice. Concentrations of DA (Panel A), DOPAC (Panel B), and the DOPAC/DA
(Panel C) in WT and CB1/CB2 KO mice given saline/vehicle, GBL (750 mg/kg)/vehicle,
or GBL/quinelorane (0.1 mg/kg) 1 h prior to decapitation, n=7-8. Columns represent
means and bars one SEM. *Significant difference between WT and CBl/CB2 KO mice,

183

DA Receptor Regulation of DA Neuronal Activity in CBI/CBZ Receptor K0 Mice

The stimulatory effects of the D2 receptor antagonist raclopride (which blocks
both pre- and post-synaptic D2 receptors) was observed by a significant increase in
DOPAC concentrations within the striatum of WT and CB1/CB2 KO mice (Figure 5-14;
Panel A). DA concentrations in the striatum were significantly decreased to a minimal,
but similar extent with raclopride treatment in both WT and CB1/C82 KO mice (Figure
5-14; Panel B). WT and CBl/CB2 KO mice also had a similar significant increase in the
DOPAC/DA ratio (Figure 5-14; Panel C).

The concentrations of DOPAC and the DOPAC/DA ratio within the nucleus
accumbens were also significantly increased to a similar extent in both WT and CB1/CB2
KO mice following raclopride treatment (Figure 5-15; Panels A & C). DA
concentrations were not Changed by raclopride treatment in WT or CBl/CB2 KO mice
(Figure 5-15; Panel B).

DOPAC concentrations within the median eminence were decreased in WT mice,
but remained unchanged in CB1/C82 KO mice (Figure 5-16; Panel A). DA
concentrations or the ratio of DOPAC/DA were not altered following raclopride in the

median eminence of either WT or CBl/CB2 KO (Figure 5-16; Panels B & C).

184

 

 

 

 

 

 

    

 

 

 

 

 

 

 

 

 

 

DOPAC
A 80 DSaline
cs” 70 i I Raclopride
'~ 60 ~
3 50 q *
g 40 a
n- 30 '
O 20 -
° 10 -
0
CB1/C82 KO
B.
DA
250 - CiSallne
" lRaclopride
at 2 .
g 00 * *
5 150 .
< 100 ‘
D
50 .
0
WT CB1/082 KO
C.
DOPAC/DA
0.50 - DSaline
a 0. 40 - * IRaciopride
2 0.0 *
5 ' '
a 0.20 ' Q
0.10 -
0.00

WT CB1/082 KO

Figure 5-14. DA receptor regulation in the striatum of WT and CB1/C82 KO mice.
Concentrations of DOPAC (Panel A) and DA (Panel B) and the DOPAC/DA ratio (Panel
C) in the striatum of WT and CB1/CB2 KO mice given saline or the D2 receptor
antagonist raclopride (1 mg/kg) 1 h prior to decapitation, n=7-8. Columns represent
means and bars one SEM. *Saline treated mice are significantly different from raclopride
treated mice, p _<_ 0.05.

185

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DOPAC
A 70 "' usaline
2’ 60 ‘ IRaclopride
2:50- * *
2 4o -
n- 30 -
8 20 -
10 -
O
CB1/682 KO
B.
DA
A200 ' USaline
5’ IRaclopride
E 150 '-
B
5
< 100 r
O
50 a
0
CB1/082 KO
C.
DOPAC/DA
1.0 - DSaline
a 0.8 J IRaclopride
2 o 6 . * *
8 .
D 0.4 *
0.2 -
0.0

 

 

 

 

 

WT CB1/682 KO

Figure 5-15. DA receptor regulation in the nucleus accumbens of WT and CBl/CB2 KO
mice. Concentrations of DOPAC (Panel A) and DA (Panel B) and the DOPAC/DA ratio
(Panel C) in the nucleus accumbens of WT and CBl/CB2 KO mice given saline or the
D2 receptor antagonist raclopride (1 mg/kg) 1 h prior to decapitation, n=7-8. Columns
represent means and bars one SEM. *Saline treated mice are significantly different fiom
raclopride treated mice, p 5 0.05.

186

DOPAC

a: DSaline
E 10 . IRaclopride

 

 

 

 

 

WT CB1/C82 KO

DA
300 - DSaline

3 250 . IRaclopride
E
E, 200 -
z 150 -
a 100 -

50 -

 

 

 

 

 

 

WT CB1/032 KO

DOPAC! DA

0.07 - DSaline
< 0.06 .. IRaclopride
3 0.05 w
E 0.04 ~
0 0.03 -
a 0.02 -
0.01 -
0.00

 

 

 

 

 

 

 

WT CB1/082 KO
Figure 5-16. DA receptor regulation in the median eminence of WT and CB1/C32 KO
mice. Concentrations of DOPAC (Panel A) and DA (Panel B) and the DOPAC/DA ratio
(Panel C) in the median eminence of WT and CB1/CB2 KO mice given saline or the D2
receptor antagonist raclopride (1 mg/kg) 1 h prior to decapitation, n=7-8. Columns
represent means and bars one SEM. *Saline treated mice are significantly different fi'om
raclopride treated mice, p 5 0.05.

187

Comparison of Serotoninergic Neuronal Function in WT and CBI/CBZ K0 Mice

5HT neurons of the raphe nuclei project axons that terminate in various regions of
the forebrain including both the striatum and nucleus accumbens (Moore etal., 1978).
5HT can facilitate the release of DA in both the striatum and nucleus accumbens and has
been implicated in the regulation of NSDA and MLDA neurons that innervate these
regions (Gudelsky and Nash, 1996; Zangen et al., 2001). 5HT concentrations are
commonly used as an index of serotoninergic neuronal terminal integrity, and the ratio of
the 5HT metabolite SHIAA to 5HT (SHIAA/5HT ratio) is considered to be the “gold
standar ” for neurochemical estimation of serotoninergic neuronal activity (Tian et al.,
1992; Darmani et al., 2003). In the present study, 5HT concentrations were compared in
the striatum and nucleus accumbens of WT and CB1/CB2 KO mice. In both brain
regions CB1/C82 KO mice had similar basal levels of 5HT compared to WT mice
(Figures 5-17; Panel A & 5-18; Panel A). The 5HT metabolite SHIAA was also
measured in both brain regions and was found not to be different in CB1/CB2 KO mice
(Figures 5-17; Panel B & 5-18; Panel B). The SHIAA/5HT ratio was similar in both WT
and CB1/C32 KO mice suggesting that the lack of CB1 and CB2 receptors does not alter
serotoninergic neuronal activity in these brain regions (Figure 5-17; Panel C & 5-18;

Panel C).

188

5HT

5HT (nglmg)

 

   

CB1/032 KO

SHIAA
1o -

6-
4i
21

SHIAA (nglmg)

 

   

CB1/C32 KO

SHIAA/5HT

1.4-
[—1.2‘
£1.0-

0.8“
i:- 0.6"
I"0.4‘

0.2 ‘

0.0-

 

 

WT CB1ICBZ KO

Figure 5-17. Concentrations of 5HT (Panel A), SHIAA (Panel B), and the SHIAA/5HT
ratio (Panel C) in the striatum of WT and CB1/C32 KO mice, n=8-10. Columns
represent means and bars one SEM.

189

N
O

_|
01

5HT (nglmg)
01 3

 

O

 

CB1/C32 KO

SHIAA

N
O

_L
01

 

SHIAA (nglmg)
8

UI

   

0

WT CB1/032 KO

SHIAA/5HT

   

WT CB1/032 KO

Figure 5-18. Concentrations of 5HT (Panel A), SHIAA (Panel B), and the SHIAA/5HT
ratio (Panel C) in the nucleus accumbens of WT and CB1/CB2 KO mice, n=8-10.
Columns represent means and bars one SEM.

190

D. Discussion

The purpose of these studies was to determine if the lack of functional CB1 and
C82 receptors in knockout mice affected central dopaminergic neuronal integrity and/or
activity, and the regulation of these neurons by pre- and post-synaptic DA receptor
mediated mechanisms. Since ECB have been implicated in their regulation three
different populations of dopaminergic neurons with cell bodies residing in either the
mesencephalon or diencephalon were studied. NSDA neurons originating in the SNpc of
the mesencephalon are regulated by ECB indirectly via ECB action at CB1 receptors
(Narushima et al., 2006; Morera-Herreras et al., 2008). MLDA neurons originating in the
VTA of the mesencephalon are regulated by GABAergic and glutamatergic neurons
possessing CB1 receptors. MLDA neuronal production of ECB inhibits pre-synaptic
neurons thus altering dopaminergic function (Melis et al., 2004b; Riegel and Lupica,
2004). TIDA neuronal activity is differentially regulated by CB depending on the
animals sex and estrogen levels at the time of CB administration, and is reflected by
alteration in prolactin concentrations (Scorticati et al., 2003). The results from the
present investigation of NSDA, MLDA, TIDA, and serotoninergic neuronal integrity and
activities in CBl/CB2 KO mice has demonstrated normal structure and functionality, and

DA receptor mediated regulation of these neuronal populations compared to WT mice.

Determining Dopaminergic Neuronal Integrity in CBI/ CB2 K0 Mice
The integrity of dopaminergic neurons was determined by comparing the
concentrations of DA in the striatum, nucleus accumbens, and median eminence of WT

and CB1/CB2 KO mice. DA concentrations in the terminal regions of dopaminergic

191

neurons reflects the amount of DA in synaptic vesicular stores, thus a change in striatum
DA concentrations could indicate a change in terminal integrity (Drolet et al., 2004). The
concentrations of DA measured in the striatum, nucleus accumbens and median eminence
were not different between WT and CB1/CB2 KO mice consistent with normal
dopaminergic terminal integrity of all three neuronal populations examined. CB1 KO
mice are known to have extracellular DA concentrations in the nucleus accumbens
comparable to WT mice (Hungund et al., 2003; Soria etal., 2005), which corroborates
the lack of difference in DA concentrations within the nucleus accumbens of WT and
CB1/CB2 KO mice.

The protein concentration of TH and DAT in the striatum can also be used as
indices of NSDA neuronal terminal integrity (Tillerson et al., 2002; J akowec et al., 2004).
TH and DAT are expressed in the axonal terminals of NSDA neurons and neurotoxin
lesioning of these terminals significantly decreases the content of both proteins within the
striatum (Storch et al., 2004). TH and DAT can therefore be considered reliable
indicators of NSDA neuronal integrity. CBl/CB2 KO mice had similar protein content
of TH and DAT compared to WT mice, correlating with similar DA concentrations
within the striatum of WT and CBl/CB2 KO mice. Taken together these results reveal
that CB1/CB2 KO mice have normal integrity of NSDA axon terminals.

The effects of CB on DA uptake by DAT have demonstrated that CB can inhibit
DAT activity (Steffens and F euerstein, 2004; Price et al., 2007). The treatment with
either a CB or a CB] receptor antagonist (AM251) reduced DA uptake by DAT in both in
vitro and in vivo settings. This would suggest that CB reduction in DAT uptake of DA is

not due to a CB1 receptor mechanism, but rather a non-CB receptor mechanism (Price et

192

al., 2007). These findings support that the lack of differential DAT expression in the
striatum of CBl/CBZ KO mice is not surprising.

NSDA neuronal cell bodies are located within the substantia nigra and are
immunoreactive for TH. Immunohistochemistry quantitative analysis of cells with TH
protein within the substantia nigra can therefore be used to identify and determine the
number of NSDA neurons. The numbers of TH immunoreactive in the substantia nigra
of WT mice were lower than those reported previously possibly due to light staining
(Liberatore et al., 1999). CB1 KO mice have typical mRNA expression of TH in the
substantia nigra (Steiner et al., 1999), which supports the findings that CB1/CB2 KO
mice have a similar number of TH neurons with the substantia nigra compared to WT
mice, and protein content of TH within the ventral midbrain. Although the number of
MLDA and TIDA neuronal cell bodies were not determined in the present study, it is
reasonable to presume that since DA concentrations were not different in either neuronal
axon terminal region, CB1/CB2 KO mice would have similar numbers of TH cells in the
ventral tegmental area and arcuate nucleus compared to WT mice. CB1 KO mice also
have similar TH mRN A levels in the ventral tegrnental area further supporting that
CB1/C32 KO mice likely have a normal number of MLDA neurons (Steiner et al., 1999).

The activity of dopaminergic neurons was determined in the present study by
measuring the DA metabolites DOPAC and HVA, and the ratio of either metabolite to
DA. The DOPAC/DA or HVA/DA ratio is reflective of DA release, reuptake, and
metabolism (Lookingland, 2005). A change in the concentrations of DOPAC, HVA, or

DA within a dopaminergic neuronal terminal region can increase or decrease the

193

DOPAC/DA or HVA/DA ratio indicating an alteration in the synthesis, release, reuptake,
and/or metabolism of DA at the neuronal terminal (Moore and Wuerthele, 1979).

CB can increase dopaminergic activity by acting at CB1 receptors in the brain
(French et al., 1997; Price et al., 2007). CB1 receptor antagonist treatment in the
presence of a CB agonist typically inhibits CB induced effects on dopaminergic neurons
(Souilhac et al., 1995; Cadogan et al., 1997; Diana et al., 1998; Gessa et al., 1998; Melis
et al., 2000; Pistis et al., 2002; Morera-Herreras et al., 2008), but by itself CB1 antagonist
treatment does not alter dopaminergic function (Gueudet et al., 1995; Cadogan et al.,
1997; Giuffrida et al., 1999; Ferrer et al., 2007). Thus, the basal activity of DA neurons
is not tonically regulated by ECB. The finding that CB1/C32 KO mice have normal
activity of dopaminergic neurons is consistent with this conclusion. Some evidence
though indicates that lack of functional CB receptors could reduce overall activity of
dopaminergic neurons since chronic administration of the CB1 receptor antagonist
rimonabant reduces activity of NSDA neurons (Gueudet et al., 1995). Nonetheless, the
results from this study support the hypothesis that CB1/CB2 KO mice have normal
NSDA, MLDA, and TIDA neuronal activity, since CB1/CB2 K0 and WT mice had
similar DOPAC and/or HV A concentrations (and DOPAC/DA and/or HVA/DA ratios) in
the striatum, nucleus accumbens, and median eminence.

Since there is a coupling between the synthesis and release of NSDA neurons, the
relative activity of these neurons can also be determined by measuring the amount of TH
protein with phosphorylated amino acid serine 4O (Bobrovskaya et al., 2007). Serine 40
is the primary serine residue on TH responsible for maximal TH activity (Dunkley et al.,

2004). Differences in serine 4O phosphorylated TH in the striatum of CB1/CB2 KO mice

194

could be indicative of differences in activity of NSDA neurons because alteration in TH
activation can affect the synthesis of DA. Protein content of serine 4O phosphorylated
TH was similar in the striatum of WT and CB1/CB2 KO mice suggesting that CB1/CB2
KO mice have comparable TH activity, contributing to a similar basal NSDA neuronal

activity.

Determining the DA Receptor Mediated Regulation of Dopaminergic Neurons in
CBI/CBZ K0 Mice

Although CB1/CB2 KO mice had normal NSDA neuronal cell number, axon
terminal integrity, and basal activity, they could still have altered DA receptor regulation
mechanisms. NSDA, MLDA, and TIDA neurons have one or more forms of feedback
inhibition including pre-synaptic short loop, post-synaptic long loop, and end product
inhibition as previously described (Chapter 1, Section G, Regulation of DA Synthesis in
Central Dopaminergic Neurons).

Pre-synaptic short loop D2 autoreceptor function can be determined by blocking
DA impulse release in the presence or absence of a D2 receptor agonist. GBL is a
precursor to y-hydroxybutyrate (GHB) a GABAB agonist that can block DA impulse
release from dopaminergic neurons (Walters and Roth, 1976; Demarest and Moore, 1979;
Nowycky and Roth, 1979; Lookingland et al., 1987b). Inhibition of DA release
following GBL administration removes feedback inhibition of DA synthesis by D2
autoreceptors on the pre-synaptic axon terminal. A lack of D2 autoreceptor stimulation
removes inhibition of TH, increases DA synthesis and vesicular and non-vesicular DA

content at the pre-synaptic axon terminal. DA short loop feedback can be maintained in

195

the presence of a D2 receptor agonist such as quinelorane. GBL treatment in the
presence of quinelorane is used commonly as a model to study short loop feedback of
dopaminergic neurons (Walters and Roth, 1976; Foreman et al., 1989; Eaton etal., 1994).

D2 autoreceptor regulation of NSDA, MLDA, and TIDA neurons was examined
in CBl/CB2 KO mice using the GBL/quinelorane model. DA and DOPAC
concentrations were increased in the striatum of WT and CB 1/CB2 KO mice treated with
GBL and this was attenuated or prevented by quinelorane treatment. The DOPAC/DA
ratio was similar in WT and CB1/C32 KO mice given GBL reflecting increased
synthesis and metabolism of DA following loss of autoreceptor inhibition of DA
synthesis. Quinelorane reduced this by maintaining autoreceptor inhibition of TH
thereby decreasing synthesis and metabolism of DA.

The maintenance of short loop D2 autoreceptor regulation in the striatum of
CB1/CB2 KO mice is not surprising since CBl receptors are not found on NSDA axon
terminals (Julian et al., 2003), and thus it is unlikely that these receptors modulate D2
pre-synaptic receptor regulation of DA synthesis. On the other hand, CB1 receptors are
found along with post-synaptic D2 receptors on striatmn medium spiny neurons (Julian et
al., 2003). CB1 and D2 receptors are known to form heterodimers on medium spiny
striatum neurons, and CB likely act as a mechanism to regulate D2 receptor induced
motor activity, since the CB decrease D2 receptor agonist mediated movement is
enhanced with the CBI antagonist rimonabant (Giuffrida et al., 1999; Ferrer et al., 2007;
Marcellino etal., 2008). Based on this information post-synaptic D2 receptor long loop

feedback would be more likely to be affected by a lack of CB] receptors.

196

 

D2 autoreceptor regulation of MLDA neurons was also evaluated in WT and
CB1/CB2 KO mice. MLDA neurons maintain short loop D2 feedback inhibition which
is even more sensitive to changes in DA concentration as compared to NSDA neurons
(Demarest et al., 1983). Surprisingly, DA concentrations were not changed with GBL
treatment in the nucleus accumbens as expected in either WT or CB1/CB2 KO mice.
DOPAC concentrations significantly decreased with GBL/quinelorane treatment in both
WT and CB 1/CB2 KO mice demonstrating that quinelorane was indeed acting at D2
autoreceptors to suppress MLDA neurons terminating in the nucleus accumbens. The
discrepancy as to why DA concentrations were not increased with GBL treatment may be
attributed to slightly greater variability in DA concentrations in the nucleus accumbens in
this study.

The DOPAC/DA ratio was significantly decreased with GBL treatment in both
WT and CB l/CB2 KO mice. These data indicate that MLDA neurons (which are more
sensitive to D2 autoreceptor regulation) (Demarest et al., 1983); are likely reducing the
activity in response to blockade of DA impulse release. Quinelorane reduced the
DOPAC/DA ratio in the nucleus accumbens to a greater extent than GBL alone
demonstrating that MLDA neurons indeed respond to short loop feedback inhibition as
previously reported (Demarest et al., 1983). Lack of firnctional CB] and CB2 receptors
in KO mice did not alter short loop feedback inhibition of MLDA neurons which would
be expected as MLDA neurons to not possess CB1 receptors (Herkenham et al., 1991b;
Melis et al., 2004a; Riegel and Lupica, 2004).

TIDA neurons lack pre-synaptic D2 receptors and thus are not regulated by D2

receptor mediated mechanisms (Demarest and Moore, 1979; Lookingland, 2005; Pappas

197

 

et al., 2008). In agreement, in the present study there was no change in DA, DOPAC, or
the DOPAC/DA ratio within the median eminence of WT and CB l/CBZ KO mice with
either GBL or GBL/quinelorane treatment. In this study CB1/CB2 KO mice had
significantly higher DA and DOPAC concentrations in the median eminence, which was
not repeated in subsequent studies (see Chapter 6) suggesting a spurious result. The
absence of CB1 and CB2 receptors does not change the inability of GBL to alter
DOPAC/DA ratio in the median eminence indicating that TIDA neurons in knockout
mice also lack autoreceptor regulation.

The present observations demonstrate that CBl/CB2 KO mice have normal D2
autoreceptor regulation in the striatum and nucleus accumbens, but do not address
whether CBl/CBZ KO mice have post-synaptic D2 receptor long loop feedback
regulatory mechanisms. Post-synaptic long loop feedback patency can be determined by
treating CB1/CB2 KO mice with the D2 receptor antagonist raclopride. Raclopride
inhibits both pro-synaptic and post-synaptic D2 receptors (Ogren et al., 1986; Eaton et al.,
1992; Eaton et al., 1994), but since CBl/CB2 KO mice have normal D2 autoreceptor
function, any differential effects seen in CB1/C32 KO mice following raclopride
treatment could be linked to changes in post-synaptic D2 receptor regulatory
mechanisms.

NSDA and MLDA neurons are both regulated by post-synaptic long loop
feedback whereas TIDA neurons are not (Eaton et al., 1992; Eaton et al., 1994; Pappas et
al., 2008). Possible differences in D2 receptor regulation in CB1/CB2 KO mice would
most likely be seen in post-synaptic D2 receptor long loop feedback, since D2 receptors

are localized in the striatum on medium spiny neurons that express CB1 receptors

198

(Martin et al., 2007). The inhibitory role CB play in dopaminergic induced activity in the
striatum (Giuffrida et al., 1999) would support the idea that a lack of CB] receptors
would affect D2 receptor regulation of NSDA neurons.

Conflicting evidence suggests that the expression and activity of D2 receptors in
the striatum of CB1/CB2 KO mice may or may not be normal since the D2 receptor is
reported to be upregulated in one CB1 KO mouse line. However, the mRNA expression
for the D2 receptor is similar to WT mice in the line of CB1 KO mice used to create the
CB1/CB2 KO mice used in these studies (Houchi etal., 2005; Gerald et al., 2006). Like
WT controls treatment of CBl/CB2 KO mice with raclopride led to a significant
reduction in DA concentrations, and an increase in DOPAC concentrations and the
DOPAC/DA ratio in the striatum all due to increased release, reuptake, and metabolism
of DA. These results indicate that a lack of CB1 and CB2 receptors does not alter long
loop post-synaptic D2 receptor feedback in the striatum of CBl/CBZ KO mice.

MLDA neurons are also regulated by D2 receptor mediated post-synaptic long
loop feedback mechanisms (Eaton et al., 1992; Pappas et al., 2008). The nucleus
accumbens contains neurons with co-localized CB1 and D2 receptors indicating a
possible post-synaptic regulatory role of CB in D2 receptor long loop regulation of
MLDA neurons (Pickel et al., 2006). The interaction of D2 and CB1 receptors could
either augment or reduce D2 receptor long loop feedback. However, in the present study
CBl/CB2 KO mice show a similar increase in DOPAC concentrations in the nucleus
accumbens with raclopride treatment and a resulting increase in the DOPAC/DA ratio

compared to WT mice. These results indicate that MLDA neurons in CB1/CB2 KO mice

199

are normally regulated by D2 post-synaptic receptors, and that a lack of functional CB1
receptors does not affect long loop feedback onto MLDA neurons.

TIDA neurons lack post-synaptic D2 receptor regulation and typically do not
exhibit any changes in DA, DOPAC, or the DOPAC/DA ratio following pharmacological
blockade of D2 receptors (Moore and Wuerthele, 1979; Berry and Gudelsky, 1991;
Pappas et al., 2008). Indeed, both WT and CB1/CB2 KO mice did not have a change in
DOPAC or DA concentrations or the DOPAC/DA ratio in the median eminence
following raclopride treatment, indicating a typical lack of post-synaptic long loop

feedback inhibition of TIDA neurons.

Serotoninergic Neuronal Integrity and Activity in CBI/CBZ K0. and WT Mice

CB are also involved in the regulation of serotoninergic neurons and like
dopaminergic neurons are influenced by CB through more than one mechanism and
results in the differential roles CB have on serotoninergic neuronal activity (Darmani et
al., 2003; Bambico et al., 2007). In the present study, the integrity and activity of
serotoninergic neurons terminating in the striatum and nucleus accumbens of WT and
CB1/CB2 mice were examined by measuring the concentrations of 5HT and SHIAA in
these regions. Neither neurochemical was different between WT and CB 1/CB2 KO mice
in the striatum and nucleus accumbens. These results are in agreement with a lack of a
role of ECB in the regulation of 5HT neurons terminating in the striatum of another line
of CB1 KO mice (Thiemann et al., 2007). CB are reported to alter the proper
development of serotoninergic neurons within the brain (Molina-Holgado et al., 1996;

Molina-Holgado etal., 1997), but the lack of a difference in concentrations of 5HT and

200

SHIAA in the striatum and nucleus accumbens of CB1/CB2 KO mice would suggest that
CB 1/CB2 KO have normal serotoninergic neuronal development.

The activity of serotoninergic neurons terminating in the striatum and nucleus
accumbens was evaluated by determining the SHIAA/5HT ratio in each brain region.

CB are reported to decrease the synthesis of 5HT in the striatum and reduce the release in
the nucleus accumbens and cortex (N akazi etal., 2000; Moranta et al., 2004; Sano et al.,
2008), and inhibition of C31 receptors increases 5HT and SHIAA concentrations in the
frontal cortex (Darmani et al., 2003; Tzavara et al., 2003). These effects on
serotoninergic neurons may be mediated by 5HT induced ECB release and feedback
inhibition on pre-synaptic CB1 receptors (Best and Regehr, 2008). In contrast to these
findings CB can also increase 5HT neuronal activity (Bambico et al., 2007). The
SHIAA/5HT ratios in the striatum and nucleus accumbens in CB1/CB2 KO mice were
similar to WT mice indicating no change in the activity of Serotoninergic neurons in
CBl/CB2 KO mice. In contrast, pharmacological blockade of CB1 receptors is reported
to increase the SHIAA/5HT ratio in the forebrain (Darmani et al., 2003). The results
presented here are in agreement with a report showing that the SHIAA/5HT ratio in the
striatum was not altered CB1 KO mice (Thiemann et al., 2007), and suggest that
CB1/CB2 KO mice have normal serotoninergic integrity and function.

The results from these studies reveal that mice lacking fimctional C81 and CB2
receptors have typical dopaminergic innervation of the striatum, nucleus accumbens, and
median eminence by NSDA, MLDA, and TIDA neurons, respectively. The activity and
DA receptor mediated regulation (or lack of in the case of TIDA neurons) are also

maintained in CB1/CB2 KO mice. Serotoninergic neurons terminating in the striatum

201

and nucleus accumbens also exhibit typical innervation and activity in CB1/CB2 KO
mice. These results suggest that the presence of CB1 or CB2 receptors is not crucial for
development of these neuronal systems or maintenance in adulthood. Lack of any
changes in these neuronal systems makes CB1/CB2 KO mice an ideal tool to determine if
the lack of ECB activity at CB1 and CB2 receptors is beneficial or detrimental in the

MPTP model of PD.

202

Chapter 6: The Effects of a Lack of Cannabinoid Receptors in the MPTP Model of
PD

A. Introduction

CB1 receptors are located throughout the basal ganglia and activation of these
receptors differentially regulates DA concentrations in the striatum depending on which
population of C31 receptors within the basal ganglia are involved (Figure 6-1)
(Herkenham et al., 1990; Herkenham et al., 1991b; Hohmann and Herkenham, 2000).

PD results in alterations in the basal ganglia function due to the loss of NSDA neurons,
including over-activation of the indirect pathway of the basal ganglia leading to inhibition
of motor output by the thalamus expressed as one of the hallmark symptoms of PD,
slowness of movement (Dauer and Przedborski, 2003). ECB levels and expression of
CB] receptors changes within the central nervous system in rodent and non-human
primate models of PD as well as in PD patients in response to a loss of NSDA neurons.
These alterations in the ECB system may contribute to the delayed development of PD
symptoms which often does not occur until 50-60% of NSDA neurons have been lost
(Dauer and Przedborski, 2003).

Expression of CB1 receptor mRNA and protein increases in the striatum of the
rodent 6-OHDA model, in non-human primates treated with MPTP, and in the brain of
humans with PD (Romero et al., 2000; Lastres-Becker et al., 2001; Gonzalez et al.,
2006). Elevation of ECB levels in the striatum of rodents and non-human primates
indicates that ECB system activity occurs with lesioning of NSDA neurons (Gubellini et
al., 2002; van der Stelt et al., 2005). The enzymatic activity of fatty acid amide hydrolase

(FAAH; which metabolizes ECB) and the uptake of ECB into cells by the anandamide

203

Cannabinoid Receptor
Expression in the Basal Ganglia

    
 

Pathway

Figure 6-1. Schematic depicting the location of CB] receptors within the basal ganglia.
CBl receptors are located on both glutamatergic (") and GABAergic (-L) terminals
within the basal ganglia. CB1 receptors are on glutamatergic cortical neurons
terminating in the striatum. Medium spiny GABAergic neurons projecting to the globus
pallidus interna (GPi), substantia nigra pars reticulata (SNpr), and globus pallidus extema
(GPe) have axonal terminal CB1 receptors. Glutamatergic neurons originating in the
subthalamic nucleus (STN) and terminating in the GPi and SNpr also have terminal CB1
receptors. ECB action at different locations within the basal ganglia can alter the activity
of the direct (excitatory) pathway or the indirect (inhibitory) pathway on the thalamus,
and also indirectly effect NSDA neurons originating in the substantia nigra pars compacta
(SNpc) and terminating in the striatum.

204

transporter (AMT) are both reduced in the striatum following 6-OHDA lesioning, thereby
contributing to increased levels of ECB (Gubellini et al., 2002). An increase in CB]
receptor expression and ECB levels in the striatum is likely a mechanism to reduce
glutamate release from corticostriatal neurons. Typically CB1 and D2 receptors are
involved in the inhibitory regulation of glutamatergic corticostriatal inputs (Gerdeman
and Lovinger, 2001; Meschler and Howlett, 2001). Loss of DA activation at D2
receptors in the striatum could explain an increase in CB1 activity in the striatum
(Gubellini et al., 2002; van der Stelt et al., 2005).

CB1 receptor expression also increases in the substantia nigra of a rodent model
of PD similar to the striatum (Gonzalez et al., 2006), and levels of the ECB 2-AG
increase in a primate MPTP model indicating ECB alterations in another portion of the
basal ganglia with NSDA neuronal loss (van der Stelt et al., 2005). The implications of
these findings is that within the SNpc region increased ECB activity could enhance the
release of DA from remaining NSDA neurons by reducing GABA release (Benarroch,
2007). In the SNpr, CB1 receptor stimulation could inhibit motor output if ECB were
acting on C3] receptors on GABAergic striatonigral neurons of the direct pathway, or
stimulate motor output via inhibition of the indirect pathway by action on glutamatergic
axonal terminals of neurons originating in the STN. Therefore, it is not known if the
enhancement of the ECB system could be beneficial or detrimental in the treatment of
PD.

The role of the ECB system within the basal ganglia in PD or models of the
disease is complex and the effects of its activation varies depending on the region of CB1

receptor activation. If ECB system activity is enhanced in PD and leads to a dampening

205

of the indirect pathway output thereby facilitating movement and increased activity of
remaining NSDA neurons, than this could be incredibly beneficial for PD patients.
However, increased activity of remaining NSDA neurons could also contribute to the
progressive loss of these neurons due to over-activity and oxidative stress. ECB
enhancement could also lead to a worsening of PD symptoms if CB action at CB1
receptors leads to increased activation of the indirect pathway. The use of CB1/C32 KO
mice therefore provides an ideal tool to explore the lack of CB receptor activity within
the basal ganglia in a model of PD. CB1/CB2 KO mice which were previously shown to
have typical integrity, activity, and DA receptor regulation of NSDA neurons will be
used along with WT mice to examine the effects of MPTP treatment, in the absence of

functional CB] and CB2 receptors.

206

Hypothesis #1: Lack of CB1 and CBZ receptors will increase MPTP induced loss of
NSDA axon terminals and cell bodies and compensatory activation of these neurons.
This hypothesis was tested by examining:

1) The concentrations of DA and TH protein content in the striatum as an index
of NSDA axon integrity following MPTP treatment.

2) The number of TH immunoreactive cells in the substantia nigra following
sub-chronic MPTP treatment.

3) The concentrations of DOPAC and HVA in the striatum and the DOPAC/DA
and HVA/DA ratios as indices of NSDA neuronal activity, and protein content
of phosphorylated serine 40 on TH.

Hypothesis #2: MPTP treatment will not alter the integrity or activity of MLDA, TIDA,
and serotoninergic neurons in CB1/CB2 KO mice following MPTP treatment compared
to WT mice.

This hypothesis will be tested by evaluating:

1) The concentrations of DA in the axon terminal regions of MLDA and TIDA

neurons to determine the integrity of neuronal terminals following MPTP

treatment.

2) The concentrations of DOPAC and HV A and the DOPAC/DA and HVA/DA

ratios to examine the activities of MLDA and TIDA neurons following MPTP

administration.

3) The concentrations of 5HT and SHIAA and the SHIAA/5HT ratio in the

striatum and nucleus accumbens to determine serotoninergic axon terminal

integrity and neuronal activity following MPTP treatment.

207

B. Materials and Methods

WT male mice obtained from (Jackson Labs) were used as controls for male
CBl/CB2 KO mice, and in CB antagonist experiments and were between 8-12 weeks of
age. All mice used for neurochemical, Western blot, or Complex I activity experiments
were sacrificed by decapitation, brains removed and immediately fiozen on dry ice
following the removal of the median eminence. Tissue was processed as previously
described in (Chapter 2, Section C, Euthanasia and Brain Tissue Preparation). The
concentrations of the neurochemicals DA, DOPAC, HVA, 5HT, and SHIAA were
determined using HPLC-EC as previously described (Chapter 2, Section D, Measurement
of Amines and Amine Metabolites in the Brain). The concentrations of MPP+ were
measured by LC-MS as previously described (Chapter 2, Section K, LC-MS Analysis of
MPP+). The content of proteins of interest (TH, DAT, alpha-synuclein, parkin, DARPP-
32, and p67Phox) was determined using Western blotting as previously described
(Chapter 2, Section G, Western Blotting). Complex I activity within striatum
mitochondrial tissue fractions was measured as previously described (Chapter 2, Section
J, Complex I Activity).

Hindbrain tissue used for immunohistochemical analysis of TH cell numbers was
dissected at the mid-coronal plane from the forebrain following decapitation, and drop
fixed in 4% paraformaldehyde for 7 days. Fixed brain tissue was sectioned in a cryostat,
stained for TH, and mounted on slides for TH cell counts of the substantia nigra using
unbiased stereology as previously described (Chapter 2, Section H, Stereology TH Cell

Counts).

208

WT and CB l/CBZ KO mice were treated with either an acute or sub-chronic
MPTP treatment paradigm to evaluate the neurotoxic effects of MPTP. Acute MPTP
treatment consisted of a single injection of saline given 4 h prior to or MPTP (10 mg/kg,
s.c.) given 1, 2, 4, or 8 h prior to decapitation. Sub-chronic MPTP treatment involved
administration of either an injection of saline or MPTP (10 mg/kg) once a day for 5 days
and mice were decapitated 3 days following the last MPTP injection. WT mice treated
with the CBI antagonist rimonabant (1 mg/kg, s.c.) and/or the CB2 antagonist SR144528
(2.5 mg/kg, s.c.) were used to determine if pharmacologic inhibition of either one or both
CB receptors attenuated the loss of NSDA axon terminals with sub-chronic MPTP
treatment. Mice were injected with either one or both CB antagonists once a day
beginning one hour prior to the first MPTP injection. CB antagonist treatment continued

once a day up to an including the morning of decapitation.

209

C. Results
Eflects of Acute MPT P Treatment on the Neurochemical Activity of NSDA Neurons in WT
and CBI/CBZ K0 Mice

The concentrations of DA and its metabolites DOPAC and HV A were measured
in the striatum of WT and CB1/CB2 KO mice treated with saline or MPTP (10 mg/kg)
and killed either 4 or 8 h later. As shown in Figure 6-2 (Panel A), saline treated WT and
CB1/CB2 KO mice had similar DA concentrations in the striatum. The concentrations of
DA significantly decreased in the striatum of both WT and CB1/C32 KO mice by 4 h
following MPTP administration. DA concentrations remained decreased 8 h after MPTP
treatment in WT mice, but began to return to levels seen with saline treatment in
CBl/CB2 KO mice (Figure 6-2; Panel A).

The concentrations of DOPAC and HVA were significantly decreased in the
striatum 4 and 8 h following MPTP treatment in both WT and CB1/CB2 KO mice
compared to saline controls (Figures 6-2; Panel B & 6-3; Panel A). The depletion of
DOPAC within the striatum was similar at 4 h in WT and CB1/C32 KO mice and
remained decreased at the 8 h time point for both (Figure 6-2; Panel B). HVA
concentrations in the striatum of WT and CBl/CB2 KO mice was significantly decreased
4 and 8 h following acute MPTP treatment, although at 4 h CB1/CB2 KO mice had a
greater reduction in HV A concentrations compared to WT mice (Figure 6-3; Panel A).

The activity of NSDA neurons in WT and CBl/CB2 KO mice was evaluated 4
and 8 h afier acute MPTP treatment by calculating the DOPAC/DA and HVA/DA ratios.
As shown in Figure 6-2 (Panel C), the DOPAC/DA ratio was not altered by MPTP

treatment 8 h later in the striatum of WT or CBl/CBZ KO mice, but by 4 h CB1/C32 KO

210

mice did have a significant decrease in the ratio. The HVA/DA ratio in the striatum of
WT mice was significantly increased 4 h following MPTP treatment compared to saline
treated mice, but returned to a value similar to saline treated mice by 8 h. The HVA/DA
ratio in CBl/CB2 KO mice was not altered following MPTP treatment compared to

saline treated mice (Figure 6-3; Panel B).

211

 

 

 

DA
-°-WT
A 250 0- CB1/CBZ KO
5’ 200
\ *
3 15° M
< 100
a 50
0 I I I 1
0 4 8
Time (hr)
DOPAC
a 40 -<>-WT
E -O-CB1ICBZ KO
a: 30
5
o 20
E
8 10
0 l l I I
4 8
Time (hr)
DOPAC/DA
g 0.20 ‘D-CB1ICBZ KO
6
g 0.15
O 0.10
O
0.05
0.00 . u u %
0 4 8
Time (hr)

Figure 6-2. Time course effects of acute MPTP treatment on DA, DOPAC and the
DOPAC/DA ratio in the striatum of WT and CB1/C82 KO mice. Mice were treated with
either saline or MPTP (10 mg/kg; s.c.) and decapitated 4 or 8 h later. Concentrations of
DA (Panel A) and DOPAC (Panel B), and the DOPAC/DA ratio (Panel C) in the striatum
of WT and CB1/CB2 KO mice, n=8-l 1. Data points represent means and bars :t one
SEM. Filled in black data points indicate a significant difference between saline treated
and MPTP treated mice within either WT or CB1/CB2 groups. *Significant difference
between WT and CB1/CB2 KO mice within a treatment group, p5 0.05.

212

 

 

 

 

HVA

25 - -o-WT
a 20 - 'G-CB1/CBZ KO
E .
a 15 " I *
5 \'
g 10 '
I 5 -

0 T I I
O 4 8
Time (hr)
B.
HVA/DA

0.25 1 -o-WT

0.20 - * Gem/032 KO
< 0.15 1
S J

0.10
>
I 0.05 «

0.00 m r .

0 4 8
Time (hr)

Figure 6-3. Time course effects of acute MPTP treatment on HVA and the HVA/DA ratio
in the striatum of WT and CB1/CB2 KO mice. Mice were treated with either saline or
MPTP (10 mg/kg; s.c.) and decapitated 4 or 8 h later. Concentrations of HVA (Panel A)
and the HVA/DA ratio (Panel B) as determined in the striatum of WT and CB1/CB2 KO
mice, n=8-1 1. Data points represent means and bars :t one SEM. Filled in black data
points indicate a significant difference between saline treated and MPTP treated mice
within either WT or CB1/CB2 groups. *Significant difference between WT and
CB1/CB2 KO mice within a treatment group, p5 0.05.

213

Expression of NSDA Axon Terminal and Striatal Proteins in WTand CBI/CBZ K0 Mice
following Acute MPT P Treatment

NSDA axon terminal integrity was evaluated by determining the protein content
of TH and DAT in the striatum of WT and CBl/CB2 KO mice treated with saline or
acute MPTP (10 mg/kg, s.c.), and normalized to cytosolic (GAPDH) and membrane
(SNAP-25) proteins, respectively. TH and DAT protein content in the striatum was
similar in saline treated WT and CB1/CB2 KO mice. As shown in Figure 6-4, acute
MPTP treatment did not alter TH or DAT content 4 or 8 h following MPTP
administration in WT or CBl/CBZ KO mice.

Alpha-synuclein protein is one of the major components in Lewy bodies, the
pathological hallmark of PD (Dauer and Przedborski, 2003). The content of alpha-
synuclein was determined in the striatum following acute MPTP treatment. As shown in
Figure 6-5 (Panels A & C), CBl/CB2 KO mice had significantly more alpha-synuclein
protein within the striatum compared to WT mice. Acute MPTP treatment did not affect
protein content of alpha-synuclein in WT mice 4 or 8 h following administration, but
CBl/CB2 KO mice showed a significant loss in alpha-synuclein in the striatum following
acute MPTP treatment at both time points (Figure 6-5; Panels A & C).

The protein content of the NADPH oxidase cytosolic subunit p67Phox associated
with microglial cells was examined in the striatum of WT and CB1/CB2 KO mice and
normalized to GAPDH as an index of microglial activation. As shown in Figure 6-5
(Panels B & C), WT and CBl/CB2 KO saline treated mice had similar levels of p67Phox
content, and acute MPTP treatment did not alter cytosolic p67Phox content in WT or

CB1/CB2 KO mice 4 or 8 h later.

214

Parkin is an E3 ligase protein associated with the ubiquitin proteasome system
which degrades and recycles damaged or mutated proteins. A mutation in the parkin
protein is associated with an autosomal recessive form of PD (Winklhofer, 2007).
Membrane bound parkin was measured in the striatrnn of WT and CB1/CB2 KO mice
treated with saline or an acute injection of MPTP. As shown in Table 6-1 and Figure 6-6,
Parkin content normalized to the membrane protein SNAP-25 was similar between saline
treated WT and CBl/CBZ KO mice and was not altered by acute MPTP treatment.

DA and CAMP regulated phosphoprotein of 32 kDa (DARPP-32) is a protein
highly expressed in medium spiny neurons of the striattun. DARPP-32 phosphorylation
is stimulated by ECB, and inhibited following pharmacological activation of D2 receptors
(Andersson et al., 2005). Phosphorylated DARPP-32 cytosolic protein content was
determined in the striatum of control and MPTP treated WT and CBl/CB2 KO mice. As
shown in Table 6-1 and Figure 6-6, phosphorylated DARPP-32 protein in the striatum
was similar between WT and CBl/CBZ KO mice treated with saline, and was not altered

by acute MPTP treatment in WT or CBl/CB2 KO mice.

215

 

 

 

 

TH
20 - -o-WT
'O'CB1/CBZ KO
15 - _
3 10 .‘r <;—;
a:
5 J
0 v v 1
o 4 8
Time (hr)
B.
DAT
5° ‘ -<>-WT
'D'CB1/CBZ KO
340 ‘ . .
E ’K
20 r
0 I I I
o 4 8
Time (hr)
C.

Saline MPTP4hr MPTPBhr
WT CBKO WT CBKO WT CBKO

THBOkDa-—->-—- unli— u-I-t III- "'—'

GAPDH...D.__.___,'-~

DAT 70 kDa ———p

Figure 6-4. Lack of an effect of acute MPTP treatment on TH and DAT protein content
in the striatum of WT and CB1/C32 KO. Mice were treated with either saline or MPTP
(10 mg/kg; s.c.) and decapitated 4 or 8 h later. Cytosolic TH protein was normalized to
GAPDH and multiplied by a factor of 100 (Panel A) and membrane bound DAT protein
was normalized to SNAP-25 and multiplied by a factor of 1,000 (Panel B) to be
expressed as relative density units (RDU), n=6-11. Representative TH, GAPDH, DAT,
and SNAP-25 protein bands for each treatment group (Panel C). Data points represent

the mean and bars i one SEM.

216

 

 

 

 

Alpha-Synuclein
150 - GWT
125 - * -<>-CB1/CBZ K0
3 100 r
g 75 - l: 3
50 .
25 -
0 I I I
o 4 8
Time (hr)
B.
p67Phox
4o - 'D'WT
30 q +CB1ICBZ KO
2
E 20 .
1o -
0 fl ‘I 1
0 4 8
Time (hr)
C.

Saline MPTP 4 hr MPTP 8 hr
WT CBKO WT CBKO WT CBKO

Alpha Synuclein 14 kDa —> .. “rs-Md» — MW

p67Phox 67 kDa —

GAPDH 36 kDa — bbbv-U

Figure 6-5. Alpha-synuclein and p67Phox cytosolic protein content in the striatum of WT
and CBl/CB2 KO mice over time following saline or MPTP treatment. Mice were treated
with either saline or MPTP (10 mg/kg; s.c.) and decapitated 4 or 8 h later. Cytosolic
alpha-synuclein protein (Panel A) and p67Phox protein (Panel B) was normalized to
GAPDH and multiplied by a factor of 1,000 to be expressed as RDU of the protein of
interest to GAPDH, n=8-11. Representative alpha-synuclein, p67Phox, and GAPDH
protein bands for each treatment group (Panel C). Data points represent the mean of each
treatment group and bars :1: one SEM. Filled in black data points indicate that MPTP
treated mice were significantly different from saline treated mice within WT or CB1/C82
KO mice. *Significant difference between WT and CBl/CB2 KO mice within a specific
treatment group, p5 0.05.

217

Table 6-1. Parkin and DARPP-32 Protein in the Striatum
Following Acute MPTP Treatment

 

 

Parkin DARPP-32
WT/ Saline 23.8 i 3.0 23.7 i 3.0
WT/ MPTP 4 hr 24.8 :1: 2.7 25.9 i 3.5
WT/ MPTP 8 hr 23.6 :1: 2.5 20.3 :1: 1.7
CB1/CB2 KO/ Saline 25.9 :1: 3.8 26.3 :t 5.0
CBl/CBZ KO/ MPTP 4 hr 26.7 :t 2.2 23.0 i 2.5
CB1/CB2 KO/ MPTP 8 hr 24.2 :1: 1.9 26.1 i 4.5

 

Table 6-1. Mice were treated with either saline or MPTP (10 mg/kg; s.c.) and decapitated
4 or 8 h later. Membrane bound parkin was normalized to SNAP-25 and multiplied by a
factor of 100, n=9-11. Cytosolic phosphorylated DARPP-32 protein was normalized to
GAPDH and multiplied by a factor of 100, n=8-11. Values displayed in the table are the
mean of each group i one SEM.

218

Saline MPTP4hr MPTPBhr
Wl' CBKO WT CBKO WT CBKO

DARRP-32 32 kDa— m t, x a. , M.’ “.J‘

GAPDH 36 kDa fib—b-ii

Figure 6-6. DARPP-32 and Parkin protein expression in the striatum in WT and
CBl/CBZ KO mice following saline or MPTP treatment. WT and CB1/CB2 KO mice
were treated with saline or acute MPTP (10 mg/kg, s.c.) and sacrificed 4 or 8 h later.
DARPP-32 protein content was normalized to the cytosolic protein GAPDH, and Parkin
the membrane protein SNAP-25. Representative cytosolic DARPP-32 and membrane
Parkin protein bands for each treatment group.

219

Acute MPT P Treatment Effects on MLDA Neurons

DA concentrations were measured in the nucleus accumbens (the axon terminal
region of MLDA neurons) in WT and CB1/C32 KO mice treated with saline or acute
MPTP (10 mg/kg, s.c.). DA concentrations in the nucleus accumbens were similar in
saline treated WT and CB1/CB2 KO mice. As shown in Figure 6-7 (Panel A), acute
MPTP treatment significantly decreased DA concentrations in the nucleus accumbens 4
and 8 h following administration in WT mice, but not CB1/CB2 KO.

The metabolites DOPAC and HVA, and the DOPAC/DA and HVA/DA ratios
were also determined in the nucleus accumbens following acute MPTP treatment in WT
and CBl/CBZ KO mice to evaluate MLDA neuronal activity. As demonstrated in Figure
6-7 (Panel B), DOPAC concentrations were significantly decreased following MPTP
treatment in the nucleus accumbens of both WT and CB1/CB2 KO mice 4 and 8 h later,
but this decrease was significantly attenuated in CB1/CB2 KO mice. HV A
concentrations were also similar in WT and CB1/CB2 KO saline treated mice, and were
not altered following acute MPTP treatment in CB1/CB2 KO mice. However, 4 h
following MPTP treatment WT mice had a significant decrease in HVA concentrations
(Figure 6-7, Panel A). The ratios of DOPAC/DA and HVA/DA in the nucleus
accumbens were examined to determine the effects of acute MPTP treatment on neuronal
activity. As shown in Figure 6-7 (Panel C), the DOPAC/DA ratio was significantly
decreased following MPTP treatment at 8 h in CBl/CB2 KO mice, but WT mice failed to
show any change in the ratio following MPTP treatment. The HVA/DA ratio was not

altered with MPTP treatment at 4 or 8 h in WT or CB1/CB2 KO (Figure 6-8; Panel B).

220

 

 

 

 

 

 

 

DA
3 150 1 'D'CBi/CBZ KO
3 *
5 100 - *
< W1;
0 50 i
0 . u a
4
Time (hr)
B.
DOPAC
’6 40 I -o-WT
% 30 . “O'CB1/CBZ KO
5 *
Q 20 j *
E 10
8 .
0 I v .
0 4 8
Time (hr)
C.
DOPAC/DA
0.5 i 'O-WT
< o 4 . O-CB1/CBZ KO
0 e
B .
E 0.3
O 0.2 r
0 0.1 -
0.0 u . .
0 4 8
Time (hr)

Figure 6-7. Time course effects of acute MPTP treatment on DA, DOPAC and the ratio
of DOPAC/DA in the nucleus accumbens of WT and CB1/CB2 KO mice. Mice were
treated with either saline or MPTP (10 mg/kg, s.c.) injection. The concentrations of DA
(Panel A) and DOPAC (Panel B), and the DOPAC/DA ratio (Panel C) in the nucleus
accumbens of WT and CB1/C32 KO mice, n=8-11. Data points represent means and
bars :t one SEM. Filled in black data points indicate a significant difference between
saline treated and MPTP treated mice within either WT or CB 1/CB2 groups.
*Significant difference between WT and CB1/CB2 KO mice within a treatment group,
p5 0.05.

221

_L _L
O 01
r J

HVA (nglmg)
U1

 

HVA

-<>-WT
-D~CB1/CBZ KO

 

0.30 -
0.25 +
3 0.20 .
2 0.15 «
i 0.10 «
0.05 -

 

Time (hr)

HVA/DA

-o-WT
-D- CB1/CB2 KO

mid

 

0.00

I

4
Time (hr)

Figure 68 Time course effects of acute MPTP treatment on HV A and the ratio of
HVA/DA in the nucleus accumbens of WT and CB1/CB2 KO mice. Mice were treated
with either saline or MPTP (10 mg/kg, s.c.) injection. The concentrations of HVA (Panel
A) and the HVA/DA ratio (Panel B) in the nucleus accumbens of WT and CB1/C32 KO
mice, n=8-11. Data points represent means and bars :1: one SEM. Filled in black data
points indicate a significant difference between saline treated and MPTP treated mice

within either WT or CB1/CB2 groups, p5 0.05.

222

Time Course Effects of MPT P Treatment on T IDA Neurons in WT and CBI/CBZ K0
Mice

The concentrations of DA were measured in the median eminence 4 and 8
following saline or acute MPTP treatment (10 mg/kg, s.c.) in WT and CB1/CB2 KO
mice. DA concentrations were similar in the median eminence of saline treated WT and
CBl/CB2 KO mice. As shown in Figure 6-9 (Panel A), acute MPTP treatment induced a
significant decrease in DA concentrations in WT mice 4 h following MPTP
administration which returned to control levels by 8 h. However, CB1/CB2 KO mice
were resistant to DA depletion in the median eminence at both 4 and 8 h following acute
MPTP treatment (Figure 6-9; Panel A).

The concentrations of DOPAC and the DOPAC/DA ratio in the median eminence
were determined to evaluate TIDA neuronal activity after acute MPTP treatment. As
shown in Figure 6-9 (Panels B & C), DOPAC concentrations and the DOPAC/DA ratio

were both significantly reduced 4 and 8 h following MPTP administration.

223

 

 

 

 

 

 

DA
200 - '°'WT
* -D-ca1ICB2 KO
3 150 -
E
a 100 - : j
5
< 50 -
o
0 . . .
0 4 8
Time (hr)
B.
DOPAC
4; 10 '1 <>'WT
g 8 . O—CB1ICBZ Ko
3
5 6 ‘
o .
g 4 9v
o 2 ‘
0 o r . .
O 4 8
Time (hr)
C.
DOPAC/DA
0.04 - (cm
O-CB1ICBZ KO
‘9! 0.03 q *
o .
g 0.02
O 0.01 -
O
0.00 I I fl
0 4 8
Time (hr)

Figure 6-9. Time course effects of acute MPTP treatment on DA, DOPAC and the ratio
of DOPAC/DA in the median eminence of WT and CBl/CB2 KO mice. Mice were
treated with either saline or an acute MPTP (10 mg/kg, s.c.) injection. The
concentrations of DA (Panel A) and DOPAC (Panel B), and the DOPAC/DA ratio (Panel
C) in the median eminence of WT and CBl/CB2 KO mice, n=8-11. Data points
represent means and bars i one SEM. Filled in black data points indicate a significant
difference between saline treated and MPTP treated mice within either WT or CB1/CB2
groups. *Significant difference between WT and CBl/CBZ KO mice within a treatment

group, p: 0.05.

224

Time Course Effects of Acute MPT P Treatment on Serotoninergic Neurons in WTand
CBI/CBZ K0 Mice

The striatum and nucleus accumbens contain serotoninergic axon terminals of
neurons originating in the dorsal and median raphe nuclei (Moore et al., 1978). The
effects of acute MPTP treatment on 5HT axon terminals and serotoninergic neuronal
activity were determined in each region. As shown in Panel A of Figures 6-10 and 6-11,
concentrations of 5HT in both the striatum and nucleus accumbens following saline
treatment were similar in WT and CB1/C32 KO mice. Acute MPTP treatment did not
alter 5HT concentrations in the striatum of either WT or CBl/CBZ KO mice, but in the
nucleus accumbens 5HT concentrations were significantly increased in CB1/C32 KO
mice with MPTP treatment (Figure 6-11; Panel A).

The concentrations of SHIAA and the SHIAA/5HT ratio were examined in both
the striatum and nucleus accumbens to determine the effects of acute MPTP treatment on
serotoninergic neuronal activity. As shown in Panel B of Figures 6-10 and 6-11, SHIAA
concentrations were not different between saline treated WT and CB1/CB2 KO mice and
were not altered following MPTP treatment in either region. However, the SHIAA/5HT
ratio indicated the serotoninergic neuronal activity was reduced following MPTP
treatment in the striatum of CB1/CB2 KO mice at 4 h and in both regions at 8 h. In
contrast, the SHIAA/5HT ratio was not altered following acute MPTP treatment in WT

mice (Figure 6-10; Panel C, & 6-11; Panel C).

225

 

 

 

 

 

 

5HT
1O - -<>-WT
A 8 . -CJ-CB1/C82 KO
3’
a 6 - H4
5 4 .
5E
an 2 ‘
0 I l I
0 4 8
Time (hr)
B.
SHIAA
10 ‘1 -<>-WT
g 3 . O—CB‘i/CBZ KO
8 6‘ gs.<$:=é
5 4‘
E 2 - .
ID
0 . . .
0 4 8
Time (hr)
C.
SHIAA/5HT
2.0 a +WT
-D-CB1ICBZ KO
l- 1.5 ‘1
a; *
3 1.0 4 (1%-<3:
35, 0.5 -
0.0 . . .
0 4 8
Time (hr)

Figure 6-10. Time course effects of acute MPTP treatment in the striatum of WT and
CB1/C32 KO mice. Mice were treated with either saline or an acute MPTP (10 mg/kg,
s.c.) injection. Concentrations of 5HT (Panel A) and SHIAA (Panel B), and the
SHIAA/5HT ratio (Panel C) in the striatum of WT and CB1/CB2 KO mice, n=7-8. Data
points represent means and bars i one SEM. Filled in black data points indicate a
significant difference between saline treated and MPTP treated mice within either WT or
CB1/CB2 groups. *Significant difference between WT and CB1/CB2 KO mice within a
treatment group, p5 0.05.

226

5HT (nglmg)

SHIAA (nglmg)

SHIAA/5HT

 

 

 

 

 

 

5HT
20 1 ‘°"WT
-D-CB1/CBZ KO
15 - *
10 I M
5 a
0 I T I
0 4 8
Time (hr)
SHIAA
10 « -<>-WT
8 . fiCBi/CBZ KO
6 ..
4 .1
2 .
0 I I I
0 4 8
Time (hr)
SHIAA/5HT
1.5 7 'O'WT
-D-CB1/CBZ KO
1.0 - *
.5. N:
0.0 . . .
0 4 8
Time (hr)

Figure 6-11. Time course effects of acute MPTP treatment in the nucleus accumbens of
WT and CB1/CB2 KO mice. Mice were treated with either saline or an acute MPTP
(10 mg/kg, s.c.) injection. Concentrations of 5HT (Panel A) and SHIAA (Panel B), and
the SHIAA/5HT ratio (Panel C) in the nucleus accumbens of WT and CBl/CB2 KO
mice, n=7-10. Data points represent means and bars i one SEM. Filled in black data
points indicate a significant difference between saline treated and MPTP treated mice
within either WT or CBl/CB2 groups. *Significant difference between WT and
CB1/C82 KO mice within a treatment group, pg 0.05.

227

Lack of Cannabinoid Receptors Partially Protects NSDA Neurons from Sub-Chronic
MPT P Neurotoxic Insult

WT and CB1/C82 KO mice were injected with MPTP using a sub-chronic
treatment paradigm (10 mg/kg; s.c. once a day for 5 days) and killed 3 days following the
last MPTP injection to determine if a lack of CB receptors would be beneficial or
detrimental with repeated neurotoxic insult to NSDA neurons. DA concentrations and
TH protein content were measured in the striatum as indices of neuronal terminal
integrity. DOPAC concentrations, the DOPAC/DA ratio, and phosphorylation of serine
40 TH was evaluated in the striatum of WT and CB1/CB2 KO mice, to determine the
effects of sub-chronic MPTP treatment on NSDA neuronal activity. The number of TH
immunoreactive cells within the substantia nigra and TH protein content in the ventral
midbrain were examined to see if MPTP treatment induced a loss of NSDA cell bodies
within the substantia nigra.

DA concentrations and TH content in the striatum were not different between
saline treated WT and CB1/CB2 KO mice. As shown in Figure 6-12 (Panel A), sub-
chronic MPTP treatment induced a significant loss in DA concentrations in the striatum
of both WT and CB1/CB2 KO mice, but the loss of DA with MPTP treatment was
attenuated in CBl/CB2 KO mice. This finding suggests that CB1/CB2 KO mice are
partially resistant to MPTP induced DA depletion. However, sub-chronic MPTP
treatment induced a significant and similar loss in TH protein in the striatum of both WT
and CB1/CB2 KO mice (Figure 6-13; Panels A & C).

DOPAC concentrations and the DOPAC/DA ratio were similar between saline

treated WT and CB1/CB2 KO mice (Figure 6-12; Panels B & C). Both WT and

228

CBl/CB2 KO mice treated sub-chronically with MPTP had a significant loss in DOPAC
concentrations in the striatum (Figure 6-12; Panel B), and the ratio of DOPAC/DA was
significantly increased in WT mice with MPTP treatment (Figure 6—12; Panel C). In
contrast, MPTP treatment did not change the ratio of DOPAC/DA in the striatum of
CBl/CB2 KO mice.

The protein content of serine 4O phosphorylated TH was also determined in the
striatum of WT and CB1/C32 KO mice treated sub-chronically with MPTP, as an
additional index of NSDA neuronal activity. Phosphorylation of serine 40 on TH can
increase with increased activity of NSDA neurons (Bobrovskaya et al., 2007). The
compensatory increase in NSDA neuronal activity demonstrated in WT mice following
MPTP treatment with the striatum DOPAC/DA ratio did not correspond with an increase
in phosphorylated serine 40 in the striatum (Figure 6-13; Panels B & C). These results
would suggest that measurement of serine 4O phosphorylation of TH protein may not be
as sensitive as the DOPAC/DA ratio to detect an increase in NSDA neuronal activity
following neurotoxin lesions.

NSDA neuronal cells were stained for TH and revealed similar morphology and
distribution within the substantia nigra of CB1/C32 KO mice treated with saline
compared to WT mice (Figure 6-14). As shown in Figure 6-15 (Panel A), comparison of
the number of NSDA neurons (as determined using an unbiased stereology cell counting
method) revealed a similar number of TH positive cells in the substantia nigra of WT and
CB1/CB2 KO mice treated with saline, which was not changed in WT or CB1/CB2 KO
mice following sub-chronic MPTP treatment. The lack of a loss in NSDA neuronal cell

bodies 3 days following the last injection of MPTP is not surprising, as a loss of cell

229

bodies with this treatment paradigm is not usually observed at 3 days following MPTP
treatment termination (Petroske et al., 2001). The protein content of TH within the
ventral midbrain containing NSDA neuronal cell bodies in the substantia nigra was
similar in saline treated WT and CB1/CB2 KO mice corresponding to a similar number
of TH immunoreactive cells in the substantia nigra (Figure 6-15; Panel B). Sub-chronic
MPTP treatment induced a small but significant loss in TH protein content within the

ventral midbrain of WT mice, which was not observed in CB1/CB2 KO mice.

230

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

   

DA
250 ' DSaIine
a 200 - IMPTP
g, 150 -
f 100 - *A
o 50 - *
0
WT CB1/C82 KO
B.
DOPAC
g 30 i USaline
a 60 - IMPTP
5
2 4O 1 ‘ *
3 20 - *
a
0
WT CB1/CB2 Ko
C.
DOPAC/DA
0.8 - DSaline
IMPTP
g 0.6 - *
a A
g 0.4 - L
8 0.2 -
0.0 -—
WT CB1/CB2 KO

Figure 6-12. Effects of sub-chronic MPTP treatment on DA, DOPAC and the ratio of
DOPAC/DA in the striatum of WT and CB1/CB2 KO mice. Mice were treated with
saline or MPTP (10 mg/kg; s.c. per day for 5 days) and decapitated 3 days later.
Concentrations of DA (Panel A) and DOPAC (Panel B), and the DOPAC/DA ratio (Panel
C) were measured in the striatum of WT and CB1/CB2 KO mice, n=9-10. Columns
represent means and bars i one SEM. *Significant difference between saline and MPTP
treated mice. "Significant difference between WT and CB1/CB2 KO mice, p _<_ 0.05.

231

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TH
20 1
DSaline
15 ‘ IMPTP
:3
a 10 *
5‘ l
O
681K282 KO
B.
Serine 40 Phosphorylated TH
5 1
4 . DSaline
IMPTP
: 3 ‘ '
a
n: 2 q
1 4
0
WT CB1/C82 KO
C.
WTI WTI CBKOI CBKOI
Saline MPTP Saline MPTP
TH 60 kDa ——§ —

Beta lll tubulin 55 kDa

Serine 40 Phosphorylated
TH 55 kDa

Beta Ill tubulin 55 kDa

—->-—--

fl

——>—--——

Figure 6-13. The effect of sub-chronic MPTP treatment on TH and phosphorylated
serine 40 TH protein in the striatum of WT and CB1/CB2 KO mice. Mice were treated
with saline or MPTP (10 mg/kg; s.c. per day for 5 days) and decapitated 3 days later. TH
or phosphorylated serine 40 TH protein was normalized to B-III tubulin and multiplied by
a factor of 100. TH protein (Panel A) and phosphorylated serine 40 TH (Panel B), and
pictorial representation of TH and phosphorylated serine 40 TH bands in WT and
CB1/CB2 KO mice (Panel C), n=6-10. Columns represent the mean and bars :1: one
SEM. *Saline treated mice are significantly different from MPTP treated mice, p 5 0.05. I

232

4x

CBKO

. q 'V.<.
:. B. . ,
. ~ ' . ‘
‘ -' 1r
, . . . .
. . . Saline
. “'5' n 1 i‘ ‘
h 7" ,.~ 0 : C"
. " as
a. . v ,"r -
7 £6
.L- ‘V‘
‘ . . I
, a , — Saline
. ,, x"
"3“" “- _ t.
F., . _ s -
. . H‘ .3: T's- I Q ‘t
s :v‘ r ..
- a ' - r
45 .-“ f .
. ' MPTP
. ‘; . 3_ a
L I '9’ (3.7 'z .
m- a r .' ~ ,
_ ,«J ‘,
r9 ‘ . .f
.1} n2 ‘3‘“; ' 5
A“ G
H. p
q»

V.

Figure 6-14. TH immunoreactive cells within the substantia nigra of WT and CB1/CB2
KO mice following sub-chronic MPTP treatment. Mice were treated with saline or
MPTP (10 mg/kg per day for 5 days) and decapitated 3 days later. WT saline (Panels A
& B), WT MPTP (Panels E & F), CBl/CB2 KO saline (Panels C & D), and CB1/CB2
KO MPTP (Panels G & H) treated mice. Panels (A, C, E, & G) were viewed at 4x, and
Panels (B, D, F, & H) at 20x. Bar below panel G is equivalent to 500 uM and bar below

panel H is 100 uM.

233

 

 

 

 

 

 

 

 

 

 

 

 

 

TH Cell Counts
t-
3 5000 - us I,"
al 8
§ 4000 ‘ IMPTP
E 3000 4 r
g 2000 -
:r: 1000 «
I'-
O -
WT CB1/CB2 Ko
B.
TH
40 - DSaline
30 .. IMPTP
8
m 20 ‘
1o -
0

WT CB1/C32 KO

Figure 6-15. Bilateral TH cell counts in the substantia nigra and ventral midbrain TH
protein content in WT and CB 1/CB2 mice treated with MPTP using the sub-chronic
treatment paradigm. Mice were treated with saline or MPTP (10 mg/kg; s.c. per day for 5
days) and decapitated 3 days later. Bilateral TH cell counts in the substantia nigra (Panel
A), n=3-7. Cytosolic ventral midbrain TH protein content normalized to B-III tubulin and
multiplied by a factor of 100 (Panel B), n=8-10. Columns represent means and bars :1: one
SEM. *Significant difference between saline and MPTP treated mice, p 5 0.05.

234

Efi’ects of Sub-Chronic MPT P Treatment on MLDA and T IDA Neurons

The integrity of MLDA axon terminals was determined by measuring the
concentrations of DA in the nucleus accumbens of WT and CB1/CB2 KO mice following
saline or sub-chronic MPTP (10 mg/kg s.c., once day or 5 days) treatment, and were
found to be similar between saline treated WT and CB1/C32 KO mice. As shown in
Figure 6-16 (Panel A), sub-chronic MPTP treatment did not alter DA concentrations in '
the nucleus accumbens demonstrating no loss of axonal integrity of MLDA neurons.

The activity of MLDA neurons was determined by measuring DOPAC
concentrations and the DOPAC/DA ratio in the nucleus accumbens. DOPAC
concentrations were similar between saline treated WT and CB1/CB2 KO mice, but sub-
chronic MPTP treatment caused a significant decrease in DOPAC concentrations in the
nucleus accumbens of CB1/C32 KO mice (Figure 6-16; Panel B). The DOPAC/DA ratio
was similar in saline treated WT and CBl/CB2 KO mice, and did not change following
MPTP treatment, suggesting that MLDA neuronal activity was not altered by MPTP
treatment (Figure 6-16; Panel C).

DA concentrations were also measured in the median eminence of WT and
CB1/CB2 KO mice following sub-chronic MPTP treatment. DA concentrations in the
median eminence were higher in CB1/C32 KO mice with saline treatment compared to
WT mice (Figure 6-17; Panel A). CB1/C32 KO (but not WT) mice had a significant
decrease in DA concentrations following MPTP treatment.

DOPAC concentrations and the DOPAC/DA ratio were also measured in the

median eminence to determine if sub-chronic MPTP treatment affected the activity of

235

TIDA neurons. DOPAC concentrations in the median eminence were similar in saline
treated WT and CBl/CB2 KO mice, and DOPAC concentrations were not changed
following MPTP treatment in WT mice (Figure 6-17; Panel B). In contrast, CBl/CB2
KO mice had a significant loss in DOPAC concentrations in the median eminence
following MPTP treatment (Figure 6-17; Panel B). However, the DOPAC/DA ratio in
the median eminence was similar between saline treated WT and CB1/C32 KO mice, and

sub—chronic MPTP treatment did not change the ratio for either (Figure 6-17; Panel C).

236

DA

DSaline
I MPTP

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

WT CB1/082 KO
B.
DOPAC
‘5 30 ‘ DSaline
g 25 ‘ IMPTP
g 20 . *
2 15 "
8 ‘2‘
D 0
WT CB1/082 KO
C.
DOPAC/DA
< 0'20 ‘ DSaline
Q 0_15 .. IMPTP
3
O
0 0.05 1
0.00

WT CB1/682 KO

Figure 6-16. Effects of sub-chronic MPTP treatment on DA, DOPAC and the ratio of
DOPAC/DA in the nucleus accumbens of WT and CBl/CBZ KO mice. Mice were
treated with saline or MPTP (10 mg/kg; s.c. per day for 5 days) and decapitated 3 days
later. Concentrations of DA (Panel A) and DOPAC (Panel B), and the DOPAC/DA ratio
(Panel C) in the nucleus accumbens of WT and CB1/C32 KO mice, n=6-10. Columns
represent means and bars i one SEM. *Significant difference between saline and MPTP

treated mice, p 5 0.05.

237

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DA
250 - DSaline
200 . * " IMPTP
E"
E 150 " ‘
:1001
a 50 -
0
WT CB1/082 KO
DOPAC
20 ' DSaline
8 IMPTP
g 15 -
g 10 - l *
o
E
o 5 I L
o
0
WT CB1/C32 KO
DOPAC/DA
0'10 ' DSaline
. IMPTP
3 0.08 N
g 0.06 1
a. 0.04 1
o
D 0.02 «
0.00
WT

238

CB1/C82 KO

Figure 6-17. Effects of sub-chronic MPTP treatment on DA, DOPAC and the ratio of
DOPAC/DA in the median eminence of WT and CBl/CB2 KO mice. Mice were treated
with saline or MPTP (10 mg/kg; s.c. per day for 5 days) and decapitated 3 days later.
Concentrations of DA (Panel A) and DOPAC (Panel B), and the DOPAC/DA ratio (Panel
C) in the median eminence of WT and CB1/C32 KO mice, n=7-10. Columns represent
means and bars i one SEM. *Significant difference between saline and MPTP treated
mice. ASignificant difference between WT and CB1/CB2 KO mice within a treatment
group, p 5 0.05.

Effects of Sub-Chronic MPT P Treatment on NSDA Neurons in WT mice Treated with
Cannabinoid Antagonists

The effects of pharmacological blockade of CB1 or CB2 receptors in the presence
of sub-chronic MPTP treatment was tested to determine if partial resistance to MPTP
induced DA depletion in the striatum of CB1/CB2 KO mice could be replicated in WT
mice. The concentrations of DA and DOPAC were measured in the striatum of WT mice
pre-treated with either a C31 antagonist or CB2 receptor antagonist prior to saline or sub-
chronic MPT P (10 mg/kg s.c., once a day for 5 days) administration. Mice were treated
with the CB1 antagonist rimonabant (1 mg/kg s.c.) or the CBZ antagonist SR14428 (2.5
mg/kg) once a day beginning the day of the first MPTP injection and continuing until the
day mice were killed, 3 days following the last MPTP injection.

DA concentrations were determined in the striatum of saline treated mice given
rimonabant, SR144528 or vehicle injections, and it was determined that CB antagonist
treatment alone did not alter DA concentrations (Figure 6-18; Panel A). As shown in
Figure 6-18 (Panel A), sub-chronic MPTP treatment induced a significant loss in striatal
DA concentrations in vehicle treated mice as compared to saline treated vehicle mice.
However, mice treated with MPTP and either rimonabant or SR144528 had a similar loss
in DA concentrations within the striatum. These results suggest that although rimonabant
and SR144528 did not alter NSDA axon terminal integrity in the presence of saline
treatment, both failed to attenuate or prevent MPTP induced loss of NSDA axon
terminals.

The activity of NSDA neurons in the presence of either rimonabant or SR144528

was also evaluated by determining DOPAC concentrations and the DOPAC/DA ratio in

239

the striatum. Rimonabant or SR144528 had no effect on NSDA activity compared to
vehicle treated mice as the DOPAC concentrations and the DOPAC/DA ratio was similar
across all treatment groups (Figure 6-18; Panels B & C). Sub-chronic MPTP treatment
induced a decrease in DOPAC concentrations and an increase in the striatum
DOPAC/DA ratio in vehicle treated mice. However, as shown in Figure 6-18 (Panels B
& C), rimonabant and SR144528 treated mice had a similar decrease in striatum DOPAC
concentrations and increase in the DOPAC/DA ratio, indicating that individual blockade
of C31 or CB2 receptors did not attenuate the compensatory increase in NSDA neuronal
activity typically seen with sub-chronic MPTP treatment.

Lack of a resistance to sub-chronic MPTP treatment in the striatum with blockade
of either CBl or CB2 receptors led to the question of whether dual blockade of both CB
receptors was required to induce resistance to DA depletion in the presence of MPTP. To
test this, WT mice were treated with saline or the sub-chronic MPTP paradigm and given
both rimonabant (1 mg/kg, s.c.) and SR144528 (2.5 mg/kg, s.c.) once a day 1 h prior to
saline or MPTP injection. CB antagonist treatment continued once daily including the
day of sacrifice.

As previously shown with individual CB receptor antagonists, dual blockade of
CB1 and CB2 receptors did not alter DA concentrations in saline treated mice, indicating
that NSDA axonal terminal integrity was not altered with CB antagonists (Figure 6-19;
Panel A). Similar to individual inhibition of CB receptors, concurrent rimonabant and
SR144528 treatment did not attenuate the loss of axon terminals caused by sub-chronic

MPTP treatment (Figure 6-19; Panel A), indicating that pharmacologic blockade of both

240

CB1 and CB2 receptors did not alter MPTP induced loss of DA in the striatum as
observed in CB1/CB2 KO mice.

The concentrations of DOPAC and the DOPAC/DA ratio were also determined to
see if dual CB receptor antagonist treatment affected the MPTP induced increase in
NSDA neuronal activity. As shown in Figure 6-19 (Panels B & C), pharmacologic
inhibition of both C31 and CBZ receptors failed to attenuate MPTP induced decrease in
DOPAC concentrations or increase in the DOPAC/DA ratio. Lack of an effect on loss of
DA in the presence of either individual or simultaneous CB1 and CBZ receptor blockade
suggests that the resistance to DA depletion seen in CB1/C82 KO mice may be due to an

unknown compensatory mechanism that occurs in CBl/CB2 KO mice.

241

 

 

 

 
 
 
 

 

    

 

 

 

 

 

 

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Figure 6-18. Lack of an effect of blockade of either CB1 or CBZ receptors on MPTP
induced changes in DA, DOPAC and the ratio of DOPAC/DA in the striatum of WT
mice. WT mice were treated with either vehicle, rimonabant (1 mg/kg; s.c.), or
SR144528 (2.5 mg/kg; s.c.) 1 h prior to either saline or MPTP (10 mg/kg; s.c. once a day
for 5 days) and continuing up until the time of decapitation, 3 days following the last
injection of MPTP. Concentrations of DA (Panel A) and DOPAC (Panel B), and the
DOPAC/DA ratio (Panel C) in the striatum, n=8-9. Columns represent means and bars :t
one SEM. *Significant difference between saline and MPT P treated mice, p 5 0.05.

242

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Figure 6-19. Lack of an effect of blockade of both CB1 and CB2 receptors on MPTP
induced changes in DA, DOPAC and the ratio of DOPAC/DA in the striatum of WT
mice. Mice were treated with vehicle or rimonabant (1 mg/kg; s.c.) and SR144528 (2.5
mg/kg; s.c.) 1 h prior to either saline or MPTP (10 mg/kg; s.c. once a day for 5 days) and
continued until 3 days following the last injection of MPT P. Concentrations of DA
(Panel A) and DOPAC (Panel B), and the DOPAC/DA ratio (Panel C) in the striatum,
n=6-8. Columns represent means and bars i one SEM. *Significant difference between

saline and MPTP treated mice, p 5 0.05 .

243

Effects ofSub-Chronic MPT P Treatment on MLDA and T IDA Neurons in WT Mice
Treated with CB Antagonists

MLDA and TIDA axon terminal integrity and neuronal activity were also
examined following either individual or dual blockade of CB1 and CB2 receptors with
rimonabant (1 mg/kg, s.c.) and/or SR144528 (2.5 mg/kg, s.c.). Mice were also treated
with either saline or a sub-chronic MPTP (10 mg/kg s.c., once a day for 5 days) as
previously described.

DA concentrations in the nucleus accumbens were not altered following CB
antagonist treatment alone or in combination in saline treated WT mice. Sub-chronic
MPTP treatment failed to alter DA concentrations except in combination with SR144528
treatment where MPTP induced a significant decrease in DA concentrations in the
nucleus accumbens (Tables 6-2 & 6-3).

DOPAC concentrations and the DOPAC/DA ratio were not altered in the nucleus
accumbens in saline treated mice given CB antagonists alone or in combination (Tables
6-2 & 6-3). DOPAC concentrations were significantly decreased in MPTP treated mice
given MPTP plus SR144528 or MPTP and both CB antagonists. Sub-chronic MPTP
treatment induced a small, but significant decrease in DOPAC concentrations (Tables 6-2
& 6-3). The DOPAC/DA ratio in the nucleus accumbens was similar across all treatment
groups, suggesting that the overall activity of MLDA neurons was not altered by these
treatments.

DA and DOPAC concentrations were similar in the median eminence in saline
treated mice administered either rimonabant and/or SR144528 (Tables 6-2 & 6-3). Sub-

chronic MPTP treatment also failed to change DA or DOPAC concentrations in the

244

presence of vehicle or either of the CB antagonists when administered individually (Table
6-2), although sub-chronic MPTP treatment did reduce DOPAC concentrations in the
presence of both CB receptor antagonists. The lack of effect on the DOPAC/DA ratio in
the median eminence indicated that CB antagonist treatment did not alter TIDA neuronal

activity (Tables 6-2 & 6-3).

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Table 6-3. Effects of Sub-chronic MPTP on DA, DOPAC and
the DOPAC ratio in the Nucleus Accumbens and Median
Eminence of Rimonabant and SR144528 Treated Mice

 

 

 

 

 

Saline/ Saline/ MPTP/ MPTP/
Vehicle Rimonabant & Vehicle Rimonabant &
SR144528 SR144528
Nucleus
Accumbens
DA 75.2 i 9.1 87.3 i 12.6 58.8 i 8.8 59.0 :1: 12.4
DOPAC 15.8 at 2.1 20.7 :t 2.8 14.5 :1: 1.5 14.0 :1: 2.3*
DOPAC/DA 0.21 d: 0.02 0.25 i 0.03 0.26 :l: 0.03 0.27 :t 0.05
Median
Eminence
DA 79.7 i 6.5 72.5 :1: 6.9 57.9 i 6.6 65.3 :L- 10.1
DOPAC 2.6 i 0.4 3.4 i 0.6 1.8 :I: 0.2 2.1 :1: 0.4*
DOPAC/DA 0.03 :1: 0.01 0.05 i 0.01 0.03 i 0.01 0.04 :2 0.01

 

Table 6-3. WT mice were treated with saline or MPTP (10 mg/kg, s.c., once a day for 5
days), and with vehicle or both rimonabant (1 mg/kg; s.c.) and SR144528 (2.5 mg/kg;

s.c) 1 hr prior to saline or MPTP administration and continuing for 3 days following the
last injection of MPTP. Concentrations of DA, DOPAC, and the DOPAC/DA ratios were
determined in the nucleus accumbens and median eminence. Values displayed in the
table are the mean of each group :t one SEM. *Significant difference between saline and
MPTP treated mice within the respective vehicle or CB antagonist treatment group.

247

Complex I Activity and MPP+ Concentrations in the Striatum of WTand CBI/CBZ K0
Mice

The partial resistance of CB1/CB2 KO mice to MPTP induced DA depletion in
the striatum could be due to differences in intrinsic mitochondrial Complex I activity or
differences in MPP+ to inhibit the activity of this enzyme. Complex 1 activity was
therefore measured in the striatum of WT and CB1/CB2 KO mice treated with saline or
MPTP (10 mg/kg, s.c.) injection and killedi4 or 8 h later. The activity of Complex I in
the striatum of WT and CB1/CB2 KO mice treated with saline was similar (Figure 6-20).
Complex I activity in the striatum following MPTP treatment was not different compared
to saline treated controls in WT or CB1/CB2 KO mice (Figure 620). These results
reveal that Complex I activity is similar between WT and CB1/CB2 KO mice, but the
assay was likely not sensitive enough too detect a reduction in Complex I activity due to
MPP+ inhibition.

Apparent partial resistance of CB 1/CB2 KO mice to MPTP could be due to
deficits in the conversion of MPTP to its bioactive metabolite MPP+. To test for this
possibility, MPP+ concentrations were measured in the striatum of WT and CB1/CB2
KO mice 1, 2, 4, or 8 h following acute MPTP (10 mg/kg, s.c.) treatment. Zero time
controls were injected with saline and killed 4 h later. As shown in Figure 6-21 (Panel
A), concentrations of MPP+ in the striatum were significantly elevated at 1, 2 and 4 h
after MPTP administration in both WT and CBl/CB2 KO mice, and by 8 h MPP+ was
almost completely eliminated. WT mice had significantly more MPP+ in the striatum at
1, 2, and 4 h following MPTP treatment as compared with CB1/CB2 KO mice. These

results indicate that although CBllCB2 KO mice can convert MPTP to MPP+ in a similar

248

manner to that of WT controls, the efficacy of this conversion is somewhat reduced in
CB1/CB2 KO mice.

DA concentrations in the striatum were also measured at 1, 2, 4, and 8 h following
MPTP administration to determine the effects of acute MPTP treatment induced vesicular
DA depletion at each of these time points. DA concentrations were similar in zero time
point saline treated WT and CB1/CB2 KO mice, and were not changed 1 or 2 h following
MPTP administration (Figure 6-21; Panel B). However, DA concentrations were
significantly decreased 4 and 8 h after MPTP treatment in WT mice, but interestingly
were not altered at these time points in CB1/CB2 KO mice (Figure 6-21; Panel B).

The DOPAC/DA ratio was also determined in the striatum at the specified time
points following acute MPTP treatment. WT mice had a significant decrease in the ratio
at 1 h with return to controls levels at 2 and 4 h, and finally increased compared to saline
controls at 8 h (Figure 6-21; Panel C). CB1/CB2 KO mice had a significant decrease 1,
2, and 4 h following MPTP administration, but by 8 h activity of NSDA neurons returned

to those of saline control animals (Figure 6-21; Panel C).

249

Complex l Activity

DWT
ICB1/CBZ KO

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Figure 6-20. Mitochondrial Complex I activity in the striatum of MPTP treated WT and
CB1/CB2 KO mice. Mice were treated either saline or an acute MPTP (10 mg/kg, s.c.)
injection and decapitated 4 or 8 h later, n=10-11. Columns represent means and bars :t
one SEM.

250

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Figure 6-21. Time course effects of a single injection of MPTP on the concentrations of
MPP+ and DA and the DOPAC/DA ratio in the striatum of WT and CB1/C32 KO mice.
WT and CB1/CB2 KO mice were injected with MPTP (10 mg/kg; s.c.) and killed at 1, 2,
4, or 8 h later. Saline treated controls served as the 0 time point and were killed at 4 h
following injection, n=5. MPP+ concentrations were measured in the striatum using LC-
MS (Panel A). Concentrations of DA (Panel B) and the DOPAC/DA ratio (Panel C) in
the striatum, n=4-5. Filled in black data points indicate a significant difference between
saline treated and MPTP treated mice within either WT or CB l/CB2 groups.
*Significant difference between WT and CB1/CB2 KO mice within a treatment group,

p: 0.05.

251

Discussion

The objective of these experiments was to determine if the presence of functional
CB1 and CB2 receptors affected MPTP induced loss of NSDA neurons. CB1/CB2 KO
mice were previously shown to have normal integrity, activity, and D2 receptor
regulation of NSDA neurons (Chapter 5), thus making CB1/CB2 KO mice an ideal tool
to investigate this topic.

The ECB system plays a role in the regulation of NSDA neurons due to the
presence of CB1 receptors throughout the basal ganglia (Herkenham et al., 1990;
Herkenham et al., 1991a; Hohmann and Herkenham, 2000). An upregulation of CB1
receptors, ECB, and changes in FAAH and AMT activity in the striatum of animal
models and/or humans indicates that ECB system is hyperactive with the loss of NSDA
neurons, especially with respect to regulation of the corticostriatal glutamatergic neurons
(Romero et al., 2000; Lastres—Becker et al., 2001; Gubellini et al., 2002; van der Stelt et
al., 2005; Gonzalez et al., 2006). An over activation of this portion of the basal ganglia
reduces glutamate release in the striatum, which is typically increased in animal models
of PD.

Upregulation in the ECB system activity in the substantia nigra in contrast could
be either beneficial or detrimental. Animal models of PD have revealed enhanced CB
receptor expression and increased ECB levels in the substantia nigra (van der Stelt et al.,
2005; Gonzalez et al., 2006). The beneficial effects of this is that increased ECB activity
in the substantia nigra pars compacta could enhance the activity of NSDA neurons and
thus increase DA release in the striatum. Although this same effect could eventually also

be detrimental if remaining NSDA neurons are over active for a long period of time,

252

which could cause a progressive loss of these neurons (F itzmaurice et al., 2003). In the
substantia nigra pars reticulata increased ECB activity could reduce motor activity if ECB
were acting on CB1 receptors on striatonigral neurons of the direct pathway (dampening
pathway activity), or alternatively enhance motor output by reduction in activity of the
indirect pathway due to CB action on axonal terminals of glutamatergic subthalamic
nucleus neurons (van der Stelt et al., 2005).

MPTP induced DA depletion in the striatum was evaluated following two
different MPTP treatment paradigms. The acute MPTP paradigm induced a decrease in
striatum DA within 4 h following a single injection of MPTP (Behrouz et al., 2007). The
decrease in DA in this model likely reflects vesicular DA depletion at 4 h, as opposed to
actual loss of NSDA neuronal integrity since there was no loss in TH or DAT protein
content in the striatum of WT mice at this time point. In contrast, the sub-chronic MPTP
model induces a significant loss in striatum DA concentrations accompanied by a loss in
TH protein content, indicating a loss of axonal integrity (Petroske et al., 2001; Drolet et
al., 2004; Shimoji et al., 2005).

Like in WT mice, acute MPTP treatment of CBl/CB2 KO mice revealed a
decrease in DA concentrations in the striatum at 4 h, but these mice had significantly
greater striatal DA concentrations at 8 h following MPTP treatment compared to WT
mice. A recovery of DA concentrations in the striatum following an acute injection of
MPTP is not apparent up to at least 32 h following administration in WT mice (Behrouz
et al., 2007). These findings indicate that MPP+ is initially inducing a similar depletion
in vesicular DA at the NSDA axon terminal presumably due to vesicular displacement of

DA from synaptic storage vesicles by MPP+ (Reinhard et al., 1987; Liu et al., 1992) in

253

both WT and CB1/CB2 KO mice. However, the mechanisms that underlie persistent
depletion in NSDA neurons terminating in the striatum of WT mice is lacking in
CB1/CB2 KO mice.

DA metabolite concentrations in the striatum were significantly decreased
following MPTP treatment consistent with a reduction in metabolism of DA, likely due to
less uptake of DA at these time points through competition with MPP+ for DAT (Barc et
al., 2002), or conversion of MPTP to MPP+ by mitochondrial MAO in NSDA axon
terminals (Takarnidoh et al., 1987). The DOPAC/DA and HVA/DA ratios indicated
variable findings in regard to activity of NSDA neurons; NSDA neurons in WT mice had
increased or lack of a change of their activity, while NSDA neurons in CB1/CB2 KO
mice had reduced or no difference in their activity following acute MPTP administration.
Since MPTP treatment alters DA metabolism through direct enzyme competitive
inhibition, changes in metabolite levels do not accurately reflect neuronal activity in this
experiment.

The recovery of MPTP induced DA depletion in CB1/CB2 KO mice in the acute
MPTP model suggests that lack of CB receptors is beneficial in an animal model of PD.
However, the acute model is not ideal to study the chronic effects of neurotoxin
administration in CB1/CB2 KO mice. Accordingly, mice were repeatedly injected with
MPTP following the sub-chronic MPTP model paradigm which induces a loss in NSDA
neuronal terminals by 3 days following the last of 5 MPTP injections (Drolet et al.,
2004)

Consistent with a previous report demonstrating that CB1 KO mice are resistant

to severe MPTP treatment (Price, 2007), results fiom the present study reveal that

254

CB1/CB2 KO mice are partially resistant to sub-chronic MPTP induced striatal DA
depletion compared to WT mice. This result was accompanied by a similar loss in
striatum TH protein in both WT and CB1/CB2 KO mice. These results suggest that
CB1/CB2 KO mice have a similar loss of NSDA axon terminals in the striatum in
response to repeated MPTP administration, but that the DA storage capacity may be
greater in the remaining unlesioned neurons in CB1/CB2 KO mice. If this is the case,
then ECB may play a role in vesicular synthesis and/or re-cycling in NSDA neurons.

The DOPAC/DA ratio in the striatum shed some light on interpretation of these
findings since this ratio is increased following sub-chronic MPTP treatment for WT mice,
but was not altered in CB1/CB2 KO mice. This would suggest that CB1/CB2 KO mice
have reduced activity of remaining NSDA neurons, which would explain a greater pool
of vesicular DA. The ability of CB agonists acting via a CB1 receptor mediated
mechanism to induce an increase in the firing of NSDA neurons and subsequent release
of DA (French et al., 1997; Melis et al., 2000; Morera-Herreras et al., 2008), would agree
with a lack of CB1 receptors leading to reduced activity of NSDA neurons. It is possible
that either a lack of or inhibition of CB1 or CB2 receptors could therefore reduce the
activity of NSDA neurons seen with MPTP treatment, thereby attenuating the progressive
loss of remaining NSDA neurons.

The number of NSDA neuronal cell bodies was also determined in the substantia
nigra along with the protein content of TH within the ventral midbrain containing the
substantia nigra. The number of TH neuronal cell bodies within the substantia nigra was
similar between saline and sub-chronic MPTP treated mice for both WT and CBl/CB2

KO, indicating that NSDA neuronal cell bodies are still intact. This corresponds to other

255

studies which have shown that MPTP does not induce a loss of NSDA neurons 3 days
post treatment (Petroske et al., 2001). The significant loss in TH observed with sub-
chronic MPTP treatment in WT mice suggests that TH protein expression may be
decreasing at this time point, but as determined by cell counts the number of NSDA
neuronal cell bodies is still intact.

The inhibition of CB1 and/or CB2 receptors using the pharmacological CB
receptor antagonists rimonabant and SR144528 in the presence of sub-chronic MPTP
treatment was done to determine which receptor type was responsible for the resistance to
MPTP induced DA depletion. The dosages of antagonists administered were chosen
based on previous studies which used these CB antagonists to effectively block CB
induced effects (Diana et al., 1998; Giuffi‘ida et al., 1999; F errer et al., 2007; Melis et al.,
2007 ; Paldyova et al., 2007; Russo et al., 2007). However, CB1 or CB2 receptor
blockade alone or in combination did not alter sub-chronic MPTP induced DA depletion.
The implications of these findings is that a lack of CB1 and CB2 receptors may not be the
underlying reason for attenuation of DA depletion in the striatum. Instead, other possible
reasons for these findings are differential ability of MPP+ to inhibit the activity of
Complex 1, a reduction in conversion of MPTP to MPP+, or epigenetic alterations in
CB1/CB2 KO mice inducing a neuroprotective mechanism.

The first possibility was addressed by measuring Complex I activity using an in
vitro assay system (J anssen et al., 2007), in striatal homogenates of WT and CB1/CB2
KO mice treated with a single injection of MPTP 4 or 8 h prior to decapitation. If
Complex I activity were initially reduced in CB1/CB2 KO mice this could explain the

resistance to MPTP induced DA depletion, because MPP+ would not be able to induce as

256

a great a reduction in enzyme activity. However, Complex I activity was similar in saline
treated WT and CB1/CB2 KO mice ruling out this possibility. Inhibition of Complex I
activity by MPP+ could not be detected 4 and 8 h following MPTP treatment, possibly
due to limited sensitivity of the assay system. Therefore, this assay system could not be
used to evaluate if there was differential inhibition of Complex I by MPP+ in CB1/CB2
KO mice.

Differential susceptibility to MPTP could be due to diminished conversion of
MPTP to MPP+ in the striatum of CB1/CB2 KO mice. MPP+ concentrations in the
striatum of WT and CB1/CB2 KO mice were determined at several time points following
MPTP administration to test for this possibility. Indeed, CB1/CB2 KO mice had lower
concentrations of MPP+ within the striatum which could explain the resistance to MPTP.
However, the reduction in MPP+ concentration in CB1/CB2 KO mice is significant but
quite small suggesting that the dramatic recovery from acute MPTP injection or
resistance to sub-chronic MPTP induced DA depletion in the striatum is likely due to
another underlying mechanism.

One possible explanation for the CB1/CB2 KO mice resistance to MPTP
treatment is an upregulation in the opioid system. Opioids like ECB can also increase
NSDA neuronal activity (Alper et al., 1980; Melis et al., 2000). The levels of mRNA for
opioid peptide precursors and activity of opioid receptors within the striatum are
increased in CB1 KO mice (Steiner et al., 1999; Uriguen et al., 2005; Gerald et al., 2006),
suggesting that an upregulation in opioid system activity could be a viable mechanism
which explains the resistance seen in CB1/CB2 KO mice. Opioid system activity is

enhanced in animal models of PD and in PD patients (Samadi et al., 2006), similar to the

257

ECB system. Opioids such as enkephalin and dynorphin, are neuropeptides which can
be released by GABAergic striatonigral or striatopallidal medium spiny neurons
projecting from the striatum, and can inhibit GABA release (Steiner and Gerfen, 1998),
also similar to the effects of ECB. These findings therefore make it plausible that
CBl/CB2 KO mice have an enhancement of opioid system activity leading to resistance
to MPTP.

The content of various proteins associated with microglial activation (p67Phox),
PD (alpha-synuclein and parkin), and crosstalk between D2 and CB receptors in the
striatum (DARPP-32) was evaluated following an acute MPTP injection. Lack of a
change in p67Phox protein content is not surprising since NSDA axonal terminals are
likely not lost this early after MPTP, but rather are depleted of DA (Reinhard et al., 1987)
(Liu et al., 1992; Behrouz et al., 2007). Surprisingly CB1/CB2 KO mice had higher basal
levels of alpha-synuclein which were decreased following MPTP treatment, whereas
alpha-synuclein was unchanged in WT mice. No relationship between ECB and alpha-
synuclein has been established, and higher basal levels of alpha-synuclein could be a
spurious finding especially since levels of this protein between WT and CBl/CB2 KO
MPTP treated mice are not different. In agreement levels of other PD-related proteins
including parkin and DARPP-32 which were not different between WT or CB1/C32 KO
mice.

The potential effects of MPTP treatment on MLDA axon terminal integrity and/or
activity in CB1/CB2 KO mice was evaluated in both the acute and sub-chronic MPT P
models. MLDA neurons are more resistant to MPTP treatment compared to NSDA

neurons, which is also seen in PD with MLDA neurons less affected than NSDA neurons

258

(U111 et al., 1985; Sundstrom et al., 1990; Behrouz et al., 2007). Interestingly, CB1/CB2
KO mice are resistant to acute MPTP induced DA depletion in the nucleus accumbens,
which was accompanied by an attenuated reduction in DOPAC concentrations. These
findings are similar to those seen in the striatum with acute MPTP treatment indicating
that dopaminergic neurons in CB1/CB2 KO mice appear to more readily recover from the
MPTP insult.

In the sub-chronic MPTP model sparing of MLDA axon terminals is more
apparent as DA concentrations are not changed in WT or CB1/CB2 KO mice. Although
DOPAC concentrations were reduced in CB1/CB2 KO mice in the nucleus accumbens,
the DOPAC/DA ratio was not altered with MPTP treatment in either WT or CB1/CB2
KO mice indicating that MLDA neuronal activity was unaffected by MPTP treatment 3
days following conclusion of MPTP treatment. CB antagonist administration concurrent
with sub-chronic MPTP treatment also failed to induce a loss in DA concentrations
except in the presence of MPTP and SR144528, although the activity of MLDA neurons
was not affected by the either CB antagonists or MPTP as indicated by the DOPAC/DA
ratio. These results would support previous finding that MLDA neurons are more
resistant to MPTP induced DA depletion than NSDA neurons (Sundstrom et al., 1990).

The effects of acute and sub-chronic treatment of MPTP on TIDA neurons were
also evaluated in WT and CB1/CB2 KO mice. Acute MPTP treatment typically induces
a transient loss in median eminence DA concentrations with recovery occurring afier a
few hours (Behrouz et al., 2007), whereas sub-chronic MPTP treatment fails to induce a
long term loss in DA concentrations (Behrouz et al., 2007). In agreement, in the present

study acute MPTP treatment of WT mice caused a transient decrease in median eminence

259

DA concentrations with recovery by 8 h, but not in CB1/CB2 KO mice. However a
decrease in DOPAC concentrations and the DOPAC/DA ratio in WT and CB1/CB2 KO
mice indicated that TIDA neurons were still affected by MPTP, but were able to maintain
normal DA concentrations in the median eminence.

Sub-chronic MPTP treatment as expected did not effect the TIDA neuron
integrity or activity. However, CBl/CB2 KO mice had significantly higher median
eminence DA concentrations than WT mice, which was likely the reason DA
concentrations in MPTP treated mice were lower. A lack of any change in the
DOPAC/DA ratio with MPTP treatment in WT or CB1/C32 KO mice is consistent with
the conclusion that higher DA concentrations in saline treated mice was likely a spurious
result. These findings would be further supported by CB antagonist treatment lacking an
effect on DA concentrations or the activity of TIDA neurons with MPTP treatment.

Serotoninergic neuronal activity was evaluated following acute MPTP treatment
in the striatum and nucleus accumbens. 5HT neurons are typically only affected long
term when exposed to very high dosing regimens of MPTP (Hallman et al., 1985; Date et
al., 1990; Rousselet et al., 2003). Interestingly, 5HT concentrations were significantly
increased in CB1/CB2 KO mice following MPTP injection in the nucleus accumbens (but
not the striatum), which was not accompanied by a change in SHIAA concentrations.
However, a significant decrease in the activity of serotoninergic neurons terminating in
both the striatum and nucleus accumbens of CB1/CB2 KO mice suggests that CB
receptors may contribute to resistance of serotoninergic neurons to MPTP reduction of

activity.

260

In conclusion, the resistance to MPTP induced DA depletion in the striatum of
CB1/CB2 KO mice in the sub-chronic model and recovery in the acute model suggested
that CB1 and/or CBZ receptors may contribute to the MPTP induced loss of NSDA axon
terminals. However, inhibition of these respective receptors individually or
simultaneously failed to pharmacologically recapitulate the resistance to MPTP seen in
CB1/CB2 KO mice. It is possible that CB1/CB2 KO mice have reduced Complex I
activity or conversion of MPTP to MPP+, but differential Complex I activity was not
responsible for the resistance and reduced MPP+ production from MPTP is not likely to
be the primary mechanism for resistance since CB1/CB2 KO had only slightly less MPP+
within the striatum following MPTP administration compared to WT mice. The
possibility exists that an enhancement in the opioid system in CB1/CB2 KO mice could
be a potential mechanism responsible for resistance to MPTP treatment, since CB1 KO
mice have increased opioid receptor activity and mRNA for opioid precursor peptides
(Steiner et al., 1999; Uriguen et al., 2005; Gerald et al., 2006).

Interestingly, CB1/CB2 KO mice were also resistant to the acute effects of MPTP
on MLDA and TIDA neurons, and serotoninergic neurons have reduced activity in the
face of this same MPTP treatment paradigm. These results suggest that MPTP does
affect several populations of neurons, but that lasting effects on these populations is

relatively limited to NSDA neurons.

261

Chapter 7: Lipopolysaccharide Activation of Microglia as an Inflammatory Model

of PD in Wildtype and CB1/CB2 Receptor KO Mice

A. Introduction

Lipopolysaccharide (LPS) can be used as a selective inflammatory model of PD
because of its inability to directly induce the death of NSDA neurons like other
neurotoxin models (Qin et al., 2004). LPS activates micro glia by binding the Toll Like
Receptor-4 (TLR-4) that frees the transcription factor nuclear factor-KB (N FKB) from a
cytoplasmic binding protein. NFKB translocates to the nucleus inducing the production
of free radical species and transcription of pro-inflammatory cytokines (Doyle and
O'Neill, 2006).

The LPS induced production of free radical species can be attributed to NFKB
activation of the enzyme NADPH oxidase which produces the ROS superoxide (Doyle
and O'Neill, 2006). Superoxide is a relatively unstable ROS incapable of crossing the
cellular membrane, but other ROS such as hydrogen peroxide and hydroxyl radical can
be formed from NADPH oxidase derived superoxide (Sumimoto et al., 2005). These
secondary ROS produced fiom superoxide have been implicated in a number of processes
including microglial proliferation, pro-inflammatory cytokine release, and induction of
iNOS leading to the production of nitric oxide (Pawate et al., 2004; Jekabsone et al.,
2006; Mander et al., 2006).

The pro-inflammatory cytokines induced by LPS directly, or indirectly through
superoxide production, include TNF-u and IL-lB. TNF-a can induce activation and

recruitment of other microglial cells to the site of inflammation (Block and Hong, 2005).

262

TNF-a can also act in an autocrine manner on receptors on the cell from which it was
released inducing further activation of NFKB which contributes to the process of
microgliosis (Kariko et al., 2004; Doyle and O'Neill, 2006). The superoxide induced
production of reactive nitrogen species such as nitric oxide in the presence of superoxide
can lead to the formation of peroxynitrite. Nitric oxide and peroxynitrite both are capable
of inhibiting the mitochondrial respiratory chain (Ebadi and Sharma, 2003).

Peroxynitrite has been implicated in NSDA neuronal death (Tieu et al., 2003), suggesting
that NADPH oxidase is a key enzyme involved in microglial derived NSDA neuronal
death induced by LPS and making LPS an ideal method to study inflammation induced
loss of NSDA neurons.

Several methods of administration of LPS in vivo have been used to induce
inflammation within the substantia nigra including direct stereotaxic injection into the
substantia nigra (intra-nigral), striatum, or peripheral injection of LPS into the peritoneal
cavity. Intra-nigral administration of LPS is ideal in that it induces a relatively localized
inflammation in the ventral midbrain that contains the highest percentage of microglial
cells in the brain (Kim et al., 2000). LPS delivered into the substantia nigra induces an
increase in the number of amoeboid activated microglia, pro-inflammatory cytokines, and
iNOS protein that leads within days to a decrease in the number of NSDA neuronal cell
bodies within the substantia nigra and DA concentrations in the striattun (Castano et al.,
1998; Gao et al., 2002b; Iravani et al., 2002; Arimoto and Bing, 2003; Zhou etal., 2005).
The percentage of NSDA neuronal cell body loss or extent of DA depletion from axon
terminals varies by length of time following injection, dosage of LPS, and whether it is

an acute injection or a chronic infusion of LPS (Castano et al., 1998; Liu et al., 2000b;

263

Gao et al., 2002b; Arai et al., 2004; Qin et al., 2004; Iravani et al., 2005). The intra-
nigral LPS induced loss of NSDA neurons appears to be rather selective since
serotoninergic neuronal terminals in the striatum appear to be only minimally affected or
not affected at all (Castano et al., 1998, 2002; Hsieh et al., 2002). The increased
vulnerability of NSDA neurons to LPS likely results from the reduced anti-oxidant ability
of these neurons (Fitzmaurice et al., 2003).

The advantage of intra-nigral injection of LPS is that the LPS stimulates nricroglia
within the cell body region of NSDA neurons in addition to increasing the number of
microglial cells within this region. However, LPS injection into the striatum can also
induce a severe loss of NSDA neuronal terminals and cell bodies which is accompanied
by an increase in iNOS in the striatum and activation of microglia in the striatum and
substantia nigra (Zhang eta1., 2006b; Hunter et al., 2007).

Unlike stereotaxic injection into the substantia nigra or striatum, peripheral
administration of LPS induces a widespread inflammatory reaction throughout the brain.
The reaction is likely initiated by cytokine stimulation from outside the blood brain
barrier, as LPS does not cross the blood brain barrier except at extremely high
concentrations (Singh and J iang, 2004; Qin et al., 2007). Peripheral LPS treatment
induces an increase in TNF-a, IL-6, and iNOS mRNA and/or protein within hours
following injection (Pitossi et al., 1997; Singh and J iang, 2004; Cunningham et al., 2005;
Qin et al., 2007). However, rapid induction of inflammation within the brain following
peripheral LPS treatment does not induce a significant decrease in the number of TH cells
until approximately 7 months following a single injection (Qin et al., 2007). This finding

does not rule out the possibility that acute inflammation will not affect dopaminergic

264

neuronal function because acute LPS treatment increases the activity of dopaminergic
neurons and TH, decreases DA in the striatum, increases the DA/DOPAC ratio in the
medial basal hypothalamus, and increases DA release into the anterior pituitary (Dunn,
1992; Cho et al., 1999; De Laurentiis et al., 2002). This fact makes the peripheral LPS
model an alternative method to study the affects of acute inflammatory reactions on
dopaminergic neurons.

An anti-inflammatory role of CB receptors has been suggested on the basis of the
observation that LPS activation of microglial cells and pro-inflammatory cytokine and
free radical species production is reduced following activation of these receptors
(Franklin and Stella, 2003; Walter et al., 2003; Carrier et al., 2004; Ehrhart et al., 2005;
Eljaschewitsch et al., 2006; Femandez-Lopez et al., 2006). However, increases in ECB
concentrations following inhibition of metabolism or blockade of reuptake increases
peripheral LPS induced plasma TNF-a concentrations more than LPS alone which was
reversed in the presence of a CB1 or a CB2 antagonist (Roche et al., 2008), suggesting
that ECB may also be pro-inflammatory.

CB] and CB2 receptors are both expressed by microglial cells in the brain,
although CB2 receptors have been implicated in most of the beneficial effects of CB
(Begg et al., 2005; Ehrhart et al., 2005). CB2 receptors are also upregulated in activated
microglial cells following LPS treatment in vitro and in vivo under neuropathological
states such as multiple sclerosis, hypoxic stroke, Alzheimer’s disease, and amyotrophic
lateral sclerosis (Benito et al., 2003; Maresz et al., 2005; Mukhopadhyay et al., 2006;

Yiangou et al., 2006; Ashton et al., 2007), and are involved in microglial migration. A

265

lack of CB receptors may or may not be beneficial for dopaminergic neurons in the face

of acute inflammation based on this information.

266

Hypothesis #1: Injection of LPS into the substantia nigra of mice will activate microglia

and induce a loss of NSDA neuronal cell bodies and axon terminals.

This hypothesis was tested by examining:
l) The concentrations of DA and DOPAC on each side of the striatum and the
DOPAC/DA ratio to evaluate the integrity of NSDA axon terminals and the
activity of NSDA neurons following LPS treatment.
2) The number of TH immunoreactive cells and the protein content of TH within
the substantia nigra unilaterally to determine if LPS induced a loss of NSDA cell
bodies.
3) Microglial NADPH oxidase activation by determining the protein content of
membrane bound p67Phox in the unilateral substantia nigra of mice injected with
LPS.

Hypothesis #2: Acute peripheral LPS treatment of WT and CB1/CB2 KO mice will alter

dopaminergic neuronal integrity or neuronal activity.

This hypothesis was tested by determining:
l) The concentrations of DA in the striatum, nucleus accumbens, and median
eminence to evaluate the integrity of NSDA, MLDA, and TIDA neuronal
terminals following LPS treatment.
2) The ratio of the concentrations of DOPAC to DA in the striatum, nucleus
accumbens, and median eminence to examine the activity of NSDA, MLDA, and

TIDA neurons.

267

B. Materials and Methods

Male C57BL/6 mice (Jackson Labs) were used for the intra-nigral LPS study and
as WT controls for CB1/CB2 KO mice for the peripheral LPS study. Male CB1/CB2 KO
mice were obtained from a breeding colony at MSU for the peripheral LPS experiment.
Intra-nigral administration of PBS vehicle or LPS (2.5, 5, or 10 ug per mouse) was done
using a stereotaxic apparatus as described (Chapter 2, Section B, Drugs). Briefly, mice
were sedated with 50 mg/kg Ketamine/2.5 mg/kg Xylazine, anesthetized with isoflurane,
and positioned in a stereotaxic apparatus. The stereotaxic coordinates at Bregma were
determined and either PBS or LPS was stereotaxically injected into the left substantia
nigra at +1.3 mm (lateral/medial plane), -3.0 mm (anterior/posterior plane), -4.7 mm
(dorsal/ventral plane) with regard to Bregma (Franklin and Paxinos, 1996). LPS was
obtained from escherichia coli 01 11:B4 (Catalog #: L3012-10 mg, Batch #: 067K4139,
Sigma Aldrich). Mice were killed either by decapitation for neurochemistry and Western
blot analysis, or perfused with 4% paraformaldehyde for immunohistochemistry analysis
of TH immunoreactive cells 14 days following Surgery as described (Chapter 2, Section
C, Brain Tissue Preparation).

Neurochemical analysis of DA and DOPAC and Western blot analysis of TH and
p67Phox protein content was done as described (Chapter 2, Section D, Assay of Amines,
Amine Metabolites, and Apocynin in Brain Tissue and Section G, Western Blot
Analysis). The brains from mice perfitsed for immunohistochemical analysis of TH
immunoreactive cells in the substantia nigra were sectioned, stained for TH, and cell
numbers counted using an unbiased stereology method as described (Chapter 2, Section

H, Immunohistochemistry and Stereology Cell Counts).

268

LPS obtained from escherichia coli 01 l 1:B4 (Catalog # L3012-5 mg, Batch #:
O47K4089, Sigma-Aldrich) was administered to WT and CB1/CB2 KO mice for the
peripheral LPS experiment. Mice received either 0.9% NaCl vehicle or LPS 5 mg/kg
body weight administered in a 0.5 mg/mL LPS solution via i.p. injection 1 h prior to
decapitation. Brains were removed following decapitation, sectioned, and prepared for
HPLC-EC analysis of DA and DOPAC in the striatum, nucleus accumbens, and median
eminence as described (Chapter 2, Section D, Assay of Amines, Amine Metabolites, and

Apocynin in Brain Tissue).

269

C. Results
Lack of an Efi’ect with Intra-nigral LPS Treatment

Mice were stereotaxically injected with either PBS vehicle or LPS (2.5, 5, or 10
ug in a volume of 0.6 uL) unilaterally into the left substantia nigra and killed 14 days
following injection of PBS or LPS. DA concentrations were measured in the striatum of
the ipsilateral and contralateral side to the injection as an index of NSDA neuronal
terminal integrity. As shown in Figure 7-1(Panel A), DA concentrations were not
significantly different between the ipsilateral and contralateral sides in mice injected with
PBS or LPS (2.5, 5, or 10 ug), indicating a lack of LPS induced axon terminal loss of
NSDA neurons with either PBS or LPS treatment.

The DOPAC concentrations and the DOPAC/DA ratio were measured unilaterally
in the striatum of PBS and LPS treated mice to determine if stereotaxic injection of PBS
or LPS altered the activity of NSDA neurons. Injection of PBS unilaterally into the
substantia nigra did not alter DOPAC concentrations or the DOPAC/DA ratio compared
to the contralateral side. Similarly LPS (2.5, 5, or 10 ug) injection did not alter DOPAC
concentrations or the DOPAC/DA ratio compared to the contralateral side indicating that
LPS did not alter the activity of NSDA neurons (Figure 7-1; Panels B and C).

NSDA neuronal cell body integrity was evaluated since LPS injection into the
substantia nigra may be more apt initially to induce a loss in cell bodies versus axon
terminals since the cell bodies of NSDA neurons were located at the site of LPS injection.
TH immunoreactive neurons within the substantia nigra were counted using the unbiased

stereology optical fi'actionater method and revealed a lack of any change in the number of

270

NSDA neuronal cell bodies within the unilateral injected substantia nigra in the presence
or absence of PBS or LPS treatment (Figure 7-2).

NSDA neuronal cell body integrity was also evaluated by examining TH protein
content in the ipsilateral and contralateral substantia nigra of PBS and LPS treated mice.
TH protein content within the substantia nigra was not changed with PBS or LPS
injection (Figure 7-3).

The protein content of the NADPH oxidase cytosolic subunit p67Phox was
measured in the substantia nigra to determine if LPS induced an increase in p67Phox
protein content in the cytosolic fraction as a consequence of microglial activation.
Surprisingly, the protein content of p67Phox was significantly increased on the ipsilateral
side of the substantia nigra of PBS treated mice. In contrast, p67Phox was not altered in
the ipsilateral side of the substantia nigra of LPS treated mice (Figure 7-4), suggesting a

lack of microglial activation with LPS intra-nigral administration.

271

DA

200 w DContralateral
Ilpsilateral

E1007
3 50i
0

LPS LPS LPS
2.5ug 5ug 10 ug

 

 

 

 

 

 

B.
DOPAC
3100 - DContralateral
E 80 . Ilpsilateral
‘2
v 60 -
0
< 40 -
8 20.
D
O ,
PBS LPS LPS LPS
2.5 ug 5 ug 10 ug
C.
DOPAC/DA
1.0 - DContralateral
B . q
:3:
o . r
0 0.2 -
0.0

 

 

 

 

 

 

 

 

PBS LPS LPS LPS
2.5ug 5ug 10ug

Figure 7-1. Ipsilateral and contralateral concentrations of DA and DOPAC and the
DOPAC/DA ratio in the striatum of mice injected unilaterally with PBS or LPS into the
substantia nigra. Mice were stereotaxically injected with either PBS vehicle or LPS 2.5,
5, or 10 ug in a volume of 0.6 uL into the left substantia nigra and decapitated 14 days
following PBS or LPS administration. Concentrations of DA (Panel A) and DOPAC
(Panel B), and the DOPAC/DA ratio (Panel C) in the contralateral and ipsilateral
striatum, n=10. Columns represent means and bars one SEM.

272

TH Cell Counts

12000 ' DContralateral
10000 - . I Ipsilateral
8000 -

6000 1
4000 -
2000 -

TH Cell Number

 

   

 

 

 

 

 

PBS LPS LPS LPS
2-5U9 5ug 10 ug

Figure 7 —2. TH cell numbers in the contralateral and ipsilateral substantia nigra of mice
injected unilaterally with either PBS or LPS into the substantia nigra. Mice were
stereotaxically injected with either PBS vehicle or LPS 2.5, 5, or 10 ug in a volume of 0.6
uL into the left substantia nigra and perfirsed 14 days following PBS or LPS
administration. The number of TH cells within each side of the substantia nigra were
counted using an unbiased stereology method, n=3-4. Columns represent means and bars
one SEM.

273

TH

30 _ L'JContralateral
I Ipsilateral

 

   

 

 

 

 

   

PBS LPS LPS LPS
2.5ug 5ug 10 ug

Figure 7-3. TH protein content in the contralateral and ipsilateral substantia nigra of mice
injected with either PBS or LPS. Mice were stereotaxically injected with either PBS
vehicle or LPS 2.5, 5, or 10 ug in a volume of 0.6 uL into the left substantia nigra and
decapitated 14 days later. TH protein content was normalized to GAPDH and multiplied
by a factor of 100 and expressed as RDU, n=10. Columns represent means and bars one
SEM.

274

p67Phox

4O - * ClContralateral
I Ipsilateral
30 -
8
a: 20 -

10-

 

 

 

 

 

 

PBS LPS LPS LPS
2.5 ug 5ug 10 ug

Figure 7-4. Cytosolic p67Phox protein content in the contralateral and ipsilateral
substantia nigra of mice injected with either PBS or LPS. Mice were stereotaxically
injected with either PBS vehicle or LPS 2.5, 5, or 10 ug in a volume of 0.6 uL into the
left substantia nigra and decapitated 14 days later. p67Phox protein content was
normalized to GAPDH and multiplied by a factor of 1,000 and expressed as RDU, n=9-
10. Columns represent means and bars one SEM. *Values for ipsilateral substantia nigra
which were significantly different from the contralateral side within a treatment group, p
< 0.05.

275

The Effects of Peripheral LPS Administration

Peripheral i.p. administration of LPS induces an acute inflammatory reaction
within the brain in 1 h, as demonstrated by an elevated cytokine profile (Qin et al., 2007).
CB reduce inflammation in brain tissue in vitro via a CB2 receptor mediated mechanism
(Ehrhart et al., 2005). The potential effects that an acute peripheral injection of LPS (5
mg/kg, i.p.) could have on dopaminergic integrity and activity was therefore examined in
WT and CB1/CB2 KO mice 1 h following LPS administration. Dopaminergic neuronal
populations evaluated included NSDA, MLDA, and TIDA neurons.

DA concentrations in the striatum were measured in WT and CBl/CB2 KO mice
injected with either saline vehicle or LPS. WT and CB1/CB2 KO mice treated with
saline vehicle had similar DA concentrations in the striatum as shown in Figure 7-5
(Panel A), and these values were not changed following LPS treatment. CB1/CB2 KO
mice treated with LPS had slightly less DA than WT mice treated with LPS. These
results indicate that acute LPS treatment does not alter NSDA axonal integrity in WT or
CB1/CB2 KO mice.

The activity of NSDA neurons was examined by determining DOPAC
concentrations and the DOPAC/DA ratio in the striatum of WT and CB1/CB2 KO mice
treated peripherally with saline vehicle or LPS. DOPAC concentrations in the striatum of
WT and CB1/CB2 KO mice treated with LPS did not change (Figure 7-5; Panel B). The
DOPAC/DA ratio was slightly higher in the striatum of CB1/CB2 KO mice treated with
saline or LPS compared to the respective treated WT group (Figure 7-5; Panel C). LPS

treatment did not alter the DOPAC/DA ratio in either WT or CB1/CB2 KO mice.

276

DA and DOPAC concentrations were also measured in the nucleus accumbens of
WT and CB1/CB2 KO mice to evaluate MLDA axon terminal integrity and activity. DA
and DOPAC concentrations were not changed with acute LPS injection within either WT
or CB1/CB2 KO mice (Figure 7-6; Panels A & B). CB1/CB2 KO mice did have elevated
concentrations of both DA and DOPAC in the nucleus accumbens in the presence or
absence of LPS compared to WT mice. The DOPAC/DA ratio was similar between WT
and CB1/C32 KO mice treated with saline, but was significantly increased in the nucleus
accumbens of WT (but not CB1/CB2 KO) mice treated with LPS (Figure 7-6; Panel C).

TIDA neuronal integrity and activity were also examined in WT and CB1/CB2
following acute LPS injection. DA and DOPAC concentrations in the median eminence
were not different in saline treated WT and CB1/CB2 KO mice (Figure 7-7; Panels A &
B). The DOPAC/DA ratio in the median eminence was also similar in WT and CB1/CB2
KO mice treated with saline, but was significantly increased by LPS in both WT and

CB1/CB2 KO mice (Figure 7-7; Panel C).

277

DA

250 q DSaline
I LPS

A

 

 

 

 

 

 

WT CB1/682 KO

DOPAC

50 l DSaline
a I LPS

 

 

 

 

 

CB1ICBZ KO

DOPAC] DA

0.30 - DSaline
< 025 . ILPS

30.20 - A A
g 0.15 -
g 0.10 -
0.05 -
0.00 -

 

 

 

 

WT CB1/C82 KO

Figure 7-5. Concentrations of DA, DOPAC, and the DOPAC/DA ratio in the striatum of
WT and CB1/CB2 KO mice following peripheral LPS administration. WT and CB1/CB2
KO mice were treated with either saline or LPS (5 mg/kg, i.p.) 1 h prior to decapitation.
Concentrations of DA (Panel A) and DOPAC (Panel B), and the DOPAC/DA ratio (Panel
C) in the striatum, n=8-9. Columns represent means and bars one SEM. "Significant
difference between WT and CB1/CB2 KO mice within a treatment group, p 5 0.05.

278

    

 

 

 

 

 

 

 

 

 

DA

200 - ElSaline
A ILPS
g’ 150 J . A A
B:
5 100 -
3

50 -
0 -—
WT CB1/C82 KO
B.
DOPAC
A 60 - DSaIine
g 50 . A A .LPS
24° 1
o 30 «
E 20 ‘
8 1o -
0 -—
CB1/082 KO
C.
DOPAC/DA

0'5 q DSaline
< 0.4 7 * ILPS
o 0.3 « T
<
3 0.2 4
o J

0.1

0.0

 

   

 

 

 

WT CB1/032 KO

Figure 7-6. Concentrations of DA, DOPAC, and the DOPAC/DA ratio in the nucleus
accumbens of WT and CB1/CB2 KO mice following peripheral LPS administration. WT
and CB1/CB2 KO mice were treated with either saline or LPS (5 mg/kg, i.p.) 1 h prior to
decapitation. Concentrations of DA (Panel A), DOPAC (Panel B), and the DOPAC/DA
ratio (Panel C) in the nucleus accumbens, n=8-9. Columns represent means and bars one
SEM. "Significant difference between WT and CB1/CB2 KO mice within a treatment
group. *Significant difference between saline and LPS treated mice, p _<_ 0.05.

279

 

 

 

 

 

 

 

 

 

 

    

 

 

 

 

DA
500 - DSaline
ILPS

E
3300 j
E.
< 200 "

100 ~

0
WT CB1/C82 KO
B.
DOPAC
DSaline
ILPS
CB1/082 KO
C.
DOPAC/DA

0-08 ‘ DSaline

0 06 * ILPS
a“ *
o .
< 0.04
8
o 0.02 «

0.00

WT CB1/C32 KO

Figure 7-7. Concentrations of DA, DOPAC, and the DOPAC/DA ratio in the median
eminence of WT and CB1/CB2 KO mice following peripheral LPS administration. WT
and CB1/CB2 KO mice were treated with either saline or LPS (5 mg/kg, i.p.) l h prior to
decapitation. Concentrations of DA (Panel A) and DOPAC (Panel B), and the
DOPAC/DA ratio (Panel C) in the median eminence, n=7-10. Columns represent means
and bars one SEM. *Significant difference between saline and LPS treated mice, p _<_

0.05.

280

D. Discussion

Intra-nigral administration of LPS has been used to study inflammatory mediated
death of NSDA neurons and typically induces microglial activation and subsequent
microglial mediated cell death of NSDA neurons (Castano et al., 1998; Gao et al., 2002b;
Iravani et al., 2002; Arimoto and Bing, 2003; Zhou etal., 2005). The purpose of
establishing this model was to use it to study potential neuroprotective anti-inflammatory
drugs, thus avoiding the direct neurotoxicity properties that other PD models have.
Results from this study where LPS (2.5, 5, or 10 ug) was unilaterally injected into the
substantia nigra demonstrated a lack of NSDA neuronal cell body or terminal loss with
LPS treatment. NSDA neuronal terminal integrity was not altered as reflected by DA
concentrations in the striatum. NSDA neuronal cell body integrity was evaluated by
determining the number of TH immunoreactive cells and total protein content of TH by
Western blotting within the substantia nigra of the ipsilateral and contralateral sides to
PBS vehicle or LPS administration. LPS treatment did not induce a loss of NSDA
neuronal cell bodies or TH protein content within the substantia nigra of the ipsilateral
side. The content of p67Phox was also measured unilaterally in the substantia nigra and
was not altered with LPS treatment compared to the contralateral side of the substantia
nigra. This finding suggests that the LPS likely did not induce activation of microglial
cells when injected directly into the ipsilateral substantia nigra. However, the cytosolic
p67Phox protein content was significantly increased with PBS administration on the
ipsilateral side of the substantia nigra. This result was likely spurious given a lack of an

increase in p67Phox on the ipsilateral side of LPS injected mice.

281'

Failure of intra-nigral LPS administration to induce microglial activation and
subsequent loss of NSDA neurons was unexpected. Studies showing that an acute intra-
nigral LPS injection induces the loss of NSDA neurons demonstrate a loss in TH
immunoreactive cells within the substantia nigra by 7 days following administration
(Kim et al., 2000; Hsieh et al., 2002; Arai et al., 2004), and mice used in this study were
killed 14 days following PBS or LPS administration. This would suggest that by 14 days
LPS should have induced a significant lesion of NSDA neurons. However, the ability of
LPS to induce inflammation can vary depending on the type of bacteria it was obtained
from and the number of endotoxin units within each mg of LPS.

The structure of LPS consists of three parts; a lipid A region which attaches to the
membrane of the bacteria, a core region linking the lipid A region to the third region, the
O-specific chain which is highly diverse (Woltmann etal., 1998; Wiese et al., 1999;
Caroff and Karibian, 2003). The biological activity of LPS is initiated by its lipid A
region with the shape of the region effecting the ability of the LPS to induce an
inflammatory reaction (Wiese et al., 1999). LPS possessing a conical shaped lipid A
region such as that obtained from escherichia coli used in these studies induces an
inflammatory reaction that is much greater than LPS having a cylindrical shape (Wiese et
al., 1999). Each batch of LPS also has a fixed number of endotoxin units/weight of LPS,
in which the higher the number of endotoxin units the greater the inflammatory reaction
should be. Although, the LPS injected into the substantia nigra and peripherally in these
studies were derived from separate batches of the same strain of escherichia coli, each
had approximately 3 million endotoxin units/mg of LPS each, as determined by Sigma-

Aldrich. Almost all studies published in the literature fail to report the number of

282

'_-_. :.I_—.__-. .___.._.—.—.— «—

endotoxin units per/weight of LPS thus making it difficult to determine the correct
dosage required to induce inflammation when using a batch of LPS that is different than
what others have used.

LPS was administered peripherally as an alternative to intra-nigral LPS
administration with the knowledge that peripheral LPS treatment at the l h time point
induces a maximal increase in TNF-o. concentrations within the brain compared to later
time points (Qin et al., 2007). The activity of dopaminergic neurons within the brain is
reported to be increased following acute peripheral LPS treatment (Dunn, 1992; De
Laurentiis et al., 2002), suggesting that acute inflammation can effect the function of
dopaminergic neurons. The ECB system is involved in the inflammatory reaction within
the brain as CB can increase or decrease the inflammatory response of microglial cells
(Ehrhart et al., 2005; Marchalanteta1., 2007b; Marchalant et al., 2007a; Roche et al.,
2008). Treatment of WT and CB1/C32 KO mice with a peripheral LPS injection did not
affect dopaminergic neuronal integrity but had differential effects on the NSDA, MLDA,
and TIDA neuronal activity.

A localized inflammatory reaction by direct intra—nigral injection of LPS typically
induces a significant inflammatory reaction and acute death of NSDA neurons versus
peripheral induced inflammation where a loss of NSDA neurons is not seen until months
later (Castano et al., 1998; Gao et al., 2002b; Iravani et al., 2002; Arimoto and Bing,
2003; Zhou et al., 2005). Peripheral LPS treatment induces a slight decrease in DA
concentrations in the striatum at 6 and 24 h following LPS administration (Cho et al.,
1999). The activity of NSDA neurons as indicated by the DOPAC/DA ratio was not

altered with LPS treatment in WT or CB1/CB2 KO mice though suggesting that if

283

peripheral LPS treatment does affect dopaminergic activity it may do so at a later time
point than at l h.

MLDA neuronal DA release and neuronal activity increases following peripheral
LPS treatment (Borowski et al., 1998; Lacosta et al., 1999) which corresponds to an
increase in the DOPAC/DA ratio in the nucleus accumbens of WT mice treated with LPS
in this study. In contrast to WT mice, CB1/CB2 KO mice did not have an increase in
MLDA neuronal activity suggesting that a lack of CB receptors reduces inflammatory
mediated effects on these neurons. Since ECB have primarily been noted for anti-
inflarnmatory effects on microglial cells this finding would contradict others, but CB can
also increase plasma concentrations of TNF-a which is believed to be the primary
mechanism by which peripheral LPS administration treatment induces brain
inflammation (Ehrhart et al., 2005; Qin et al., 2007; Roche et al., 2008). An increase in
TNF-a concentrations that is reversed in the presence of CB1 or CB2 antagonists would
instead support the findings that, CB1/CB2 KO mice do not have an increase in MLDA
neuronal activity with LPS treatment.

LPS induced inflammation induces a significant increase in the activity of TIDA
neurons as indicated by an increase in the DOPAC/DA ratio in the median eminence
(Lacosta et al., 1999; De Laurentiis et al., 2002). The release of the hormone prolactin is
reduced following LPS treatment and may be due to an increase in TIDA neuronal
activity since TIDA neurons inhibit the release of prolactin (De Laurentiis et al., 2002;
Hollis et al., 2005). TIDA neuronal activity significantly increased with LPS in WT and
CB1/CB2 KO mice, which is in agreement with previous findings (De Laurentiis et al.,

2002). TIDA axon terminals reside outside of the blood brain barrier. Therefore, it is

284

possible that a peripheral inflammatory reaction may increase the activity of these
neurons, whereas inflammatory mediators must cross the blood brain barrier in order to
change the activity of NSDA neurons that completely reside within the brain.

In summary the lack of a loss in NSDA neuronal cells with intra-nigral LPS was
surprising as intra-nigral LPS administration is reported to induce a loss of NSDA
neuronal cell bodies and terminals over time. It is possible though that either the LPS
used in this study did not successfully activate microglial cells or that the number of
endotoxin units injected was to low to induce inflammation mediated loss of NSDA
neurons. The use of the peripheral LPS model instead facilitated the investigation as to if
acute inflammation affected dopaminergic neuronal function in the presence or absence
of CB1 or CB2 receptors. CB1/CB2 KO mice indeed had either no change in activity
with LPS treatment (MLDA neurons) in contrast to WT mice, or an increase in TIDA
neuronal activity like WT mice with LPS peripheral administration. These results
suggest that CB receptors may play a role in acute inflammation effects on dopaminergic

neuronal activity.

285

 

Chapter 8: Concluding Remarks

PD is a debilitating neurodegenerative disease associated with the progressive loss
of NSDA neurons. The progressive nature of DA neuronal loss allows remaining NSDA
neurons to compensate for the loss of others, and explains the delayed diagnoses of PD
which occurs generally when approximately 50-60% of NSDA neurons and 80% of DA
has been depleted. PD is likely due to multiple causes of NSDA neuronal loss which
include, but are not limited to; exposure to environmental toxins, mitochondrial
dysfunction, oxidative stress, protein misfolding and aggregation, and inflammatory
related damage to NSDA neurons.

The location of NSDA neurons makes these neurons especially vulnerable to
oxidative stress and inflammation. These neurons are susceptible to increased
endogenous oxidative stress due to oxidation of cytosolic DA to form DA quinones that
bind and damage cellular proteins. NSDA neurons also contain iron which reacts with
hydrogen peroxide to form ROS hydroxyl radicals which are highly reactive. When
NSDA neurons die due to increased oxidative stress the cellular debris activates
microglial cells within the SNpc. The ventral midbrain containing the SNpc has the
highest number of microglia compared to other brain regions likely making this region
more prone to an intense inflammatory reaction following loss of NSDA neurons. Once
initiated, the inflammatory reaction can be self-amplifying leading to microgliosis.
Microglial activation persists within the SNpc in PD patients and several animal models
of the disease long after neurotoxin or inflammatory insult.

The challenge of developing neuroprotective therapies for PD has mainly focused

on treatments that could either slow or halt the loss of remaining unlesioned NSDA

286

 

neurons. These targets include reducing oxidative stress and inflammation by inhibiting
the production of free radical species and pro-inflammatory cytokines which continually
induce the production of one another. Alternatively potential treatments to regenerate or
replace NSDA neurons have been attempted to restore function of the NSDA neurons.

The purpose of this dissertation was to identify one or more potential
neuroprotective treatments for PD using neurotoxic and inflammatory models of the
disease to test pharmacologic agents or explore the effects of the genetic knockout of
CB1 and CB2 receptors. Although the effects of MPTP and LPS on other neuronal
populations in the presence or absence of these various neuroprotective drugs were also
examined they were not the primary focus of this dissertation and thus will not be
discussed in this chapter.

The use of MPTP and LPS under differing administration paradigms was aimed at
evaluating potential neuroprotective effects that may be observed depending on the cause
of PD. Acute MPTP treatment was used to examine the effects of rapid DA depletion
and is ideal to evaluate NSDA neuronal activity in response to a neurotoxic insult. The
repeated acute MPTP model was used to evaluate the effects of induction of microglial
activation in response to necrotic loss of NSDA neurons. The sub-chronic and chronic
MPTP administration paradigms were aimed at examining neuroprotective potential in a
long term repetitive lesioning of NSDA neurons and microglial activation.

The intra-nigral LPS model was used to study a localized inflammatory mediated
death of NSDA neurons, although failure of inflammation to be observed in this model
prevented it fi'om being used in subsequent neuroprotection studies. The peripheral LPS

model was used to study the acute effects of inflammation on NSDA neurons, rather than

287

,.,.. L L1 '__‘

loss of NSDA neurons since mice were killed within 1 h following administration of
LPS. The differences in these models and treatment paradigms demonstrates their utility
to study mechanisms of NSDA neuronal degeneration or activity, but also reveals that
none of these models can replicate the exact pathology that occurs with PD in humans.

The first objective of this dissertation investigated the potential induction of
neurogenesis of adult neural stem cells by increasing cGMP in the brain resulting from
Sildenafil inhibition of phosphodiesterase-5 (PDE5). Three different Sildenafil
administration paradigms were employed to determine if Sildenafil was neuroprotective
in the chronic MPTP model. The chronic MPTP model was ideal for this study because
it consists of a long term repeated exposure of the neurotoxin, which was extended by
concurrent administration with probenecid.

Neuroprotection was examined by determining if there was a potential recovery in
NSDA neuronal cell body or axon terminal loss with Sildenafil treatment in the presence
of MPTP, and if this was due to the migration and formation of new NSDA neurons from
adult progenitor stem cells. Previous studies in a rodent stroke model strongly suggested
that Sildenafil would be neuroprotective, since it resulted in partial neurologic recovery.
However, since Sildenafil failed to cause generation of new NSDA neurons it was
concluded that this compound was not neuroprotective in this MPTP model. However,
one key finding resulting fiom these studies is that Sildenafil did not induce the loss of
NSDA neurons itself in the presence of either saline or MPTP treatment. This finding
suggests that patients with PD can safely take Sildenafil as a treatment for erectile
dysfunction (ED). The greater incidence of ED in patients with PD makes this finding

especially important.

288

The second objective of this dissertation investigated the NADPH oxidase
enzyme inhibitor apocynin as a potential neurOprotective agent in the repeated acute and
sub-chronic MPTP inflammatory models of PD. The contribution of microglial NADPH
oxidase to MPTP induced loss of NSDA neurons following the initial toxic insult is clear
as loss of NSDA neurons is attenuated in mice lacking the crucial gp91Phox subunit of
the enzyme. Apocynin appeared initially to be an ideal agent to reduce the loss of NSDA
neurons, but the short half-life of this drug made it hard to establish the fiequency and
dosage required to maintain continual inhibition of NADPH oxidase. The choice to use
two different MPTP treatment paradigms was an attempt to first observe a potential
neuroprotective effect in a model that has a slightly more modest loss of NSDA neurons
and likely less microglial activation, although NSDA axon terminal loss was not
attenuated in this model with apocynin. The repeated acute model was employed to
induce not only a more severe and rapid loss of NSDA neurons but also a greater
activation of microglial cells and NADPH oxidase. However, even in the repeated acute
MPTP model NSDA neuronal loss or NADPH oxidase activation was not reduced by
apocynin. These results demonstrated the challenges of working with a drug that has a
short half-life and that has a suspected active metabolite diapocynin which has only
minimally been studied.

The role of the ECB system and CB1 and CB2 receptors were studied as a final
objective of this dissertation. Initially, NSDA neuronal integrity, activity, and regulation
were investigated to determine if CBl/CB2 KO mice were different compared to WT
mice. Results indicated that CB1/CB2 KO mice were similar to their WT counterparts

and were deemed ideal to study how the lack of CB receptors may be beneficial or

289

detrimental in acute or sub-chronic MPTP DA depletion and or loss of NSDA neurons. It
was hypothesized that CB1/CB2 receptor KO mice would be more prone to MPTP
induced DA depletion in the striatum, since the ECB appears to be upregulated in animal
models of PD, likely leading to enhancement of dopamine release.

Results from these studies instead led to the rejection of the proposed hypothesis
as CB1/CB2 KO receptors were partially resistant to MPTP induced DA depletion in the
striatum of the sub-chronic model, and recovered from DA depletion in the acute model.
Surprisingly, attenuation in DA depletion was not accompanied by less of a reduction in
TH protein in the striatum, suggesting that CB1/CB2 KO mice had a similar loss of
NSDA neuronal terminals. This finding contradicts resistance to DA depletion but lack
of an increase in NSDA activity in CB1/C32 KO mice suggested that lack of CB
receptors led to less depletion of vesicular DA explaining the differential results between
DA and TH loss in the striatum. The ability of CB agonists to increase firing of NSDA
neurons and release of DA would therefore support these results.

The implications of these findings is that CB1 and/or CB2 receptor inhibition
could potentially be used to reduce the compensatory activity of remaining NSDA
neurons, thereby attenuating the progressive loss of these neurons. However, either
individual or simultaneous inhibition of CB1 and CB2 receptors failed to replicate
findings of resistance to DA depletion in the sub-chronic MPTP model observed in the
striatum of CB1/CB2 KO mice. Based on these results it was hypothesized that either
CB1/CB2 KO mice had differences in Complex I activity or MPP+ inhibition of the
Complex, reduced conversion of MPTP to MPP+, or a genetic compensation due to a

lack of CB1 and CB2 receptors which conferred the resistance to DA depletion.

290

Measurement of Complex I activity revealed CB1/CB2 KO mice had typical
activity of the complex, but CB1/CB2 KO mice did have less MPP+ in the striatum at 1,
2, and 4 h following an acute injection of MPTP. This would suggest that CB1/CB2 KO
mice had reduced metabolism of MPTP to MPP+, but the decrease in MPP+
concentrations in the striatum compared to WT mice was so small that it is highly
unlikely that this was the major reason why CB1/CB2 KO mice were partially resistant to
MPTP. Thus, future studies to determine the reason for the resistance observed in
CB1/CB2 KO mice would need to focus on genetic differences in the CB1/CB2 KO
mice. Investigation into the opioid system involved in the regulation of NSDA neurons is
one potential target to focus on as CB1 KO mice have higher mRNA levels of opioid
peptide precursors.

The role of CB in inflammation within the brain has mainly been noted to be anti—
inflammatory in nature. This was investigated in these studies using the acute peripheral
LPS model in WT and CB1/CB2 KO mice to determine the acute effects of inflammation
on NSDA neurons. NSDA neuronal activity was not affected in WT or CB1/CB2 KO
mice with LPS, suggesting that lack of CB1 and CB2 receptors at least with an acute
inflammatory reaction is not detrimental to these neurons.

As the work of this dissertation has demonstrated the search and investigation for
potential neuroprotective treatments for PD will be an ongoing and challenging one.
Although Sildenafil, did not lead to the generation of new NSDA neurons it can not be
ruled out that other PDE inhibitors may be neuroprotective, since the levels of other PDE
may be higher in the brain then PDES and thus contribute to a greater increase in cGMP

to induce neurogenesis.

291

The over arching conclusion for apocynin as a potential neuroprotective drug is
that the pharmacokinetics of the drug made it hard to determine the ideal time points,
frequency and methods of administration. Apocynin or another NADPH oxidase
inhibitor may still be determined to be neuroprotective in the future if the
pharmacokinetics of the drug is better known. Additionally, the use of another PD model
that is solely inflammatory mediated may have been more apt to reveal a neuroprotective
effect of apocynin, as opposed to a MPTP model which induces indirect microglial
activation in response to the loss of NSDA neurons.

Interestingly, mice lacking CB1 and CB2 receptors were partially resistant to
MPTP induced DA depletion in the striatum, that is likely due to reduced compensatory
activity of intact NSDA neurons. Increased activation of NSDA neurons following
neurotoxin lesioning contributes to the progressive loss of these remaining neurons, so if
a CB antagonist could be given to slow or prevent the loss of remaining neurons this
would be a potential neuroprotective drug. However, failure of either CB1 or CB2
receptor antagonists to prevent the loss of DA with sub-chronic MPTP treatment argued
against this hypothesis. Differential inhibition of Complex I was not the primary
mechanism for the resistance observed in CB1/CB2 KO, suggesting that a developmental
compensation in CB1/CB2 KO mice due to the absence of functional CB receptors may
be responsible for this resistance. Future experiments would need to explore the integrity
and activity of other systems that are involved in regulation of NSDA neurons in the
CB1/CB2 KO mice. The opioid system which is also involved in regulating NSDA
neurons would be the first ideal target to investigate this hypothesis since CB1 KO mice

appear to have increased opioid peptide precursor mRN A.

292

In summary the objectives of this dissertation to identify one or more potential
neuroprotective treatments for PD proved challenging. Although Sildenafil and apocynin
were not neuroprotective some conclusions were still made from these studies. However,
most interesting were the resistance of CB1/CB2 KO mice to MPTP treatment, these
studies demonstrated that even if CB receptors are not the primary reason for this

resistance further characterization of the these mice may still identify this reason.

293

 

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