Metabotropic glutamate and adenosine receptors in the monkey striatopallidal complex and their involvement in Parkinsonism: Anatomy, physiology, and behavior Public
Bogenpohl, James William (2013)
Abstract
Long term dopamine replacement therapies for Parkinson's disease (PD) induce debilitating side effects. The discovery of non-dopaminergic pharmacotherapeutics for PD which could replace or potentiate dopaminergic drugs represents a strategy whereby side effects could be reduced. In this thesis, anatomical, electrophysiological, and behavioral properties of three potential non-dopaminergic drug targets in the striatopallidal complex were examined. First, we demonstrated that the metabotropic glutamate receptor 5 (mGluR5) antagonist 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) potentiates the antiparkinsonian effect of low doses of the D2-like dopamine receptor (D2LR) agonist pramipexole in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. This combination treatment strategy may be able to reduce the development of side effects commonly associated with high doses of D2LR agonists in PD. The adenosine A2A receptor (A2AR) is another attractive non-dopaminergic target for PD pharmacotherapy. In rodents, synergistic antiparkinsonian effects of A2AR and mGluR5 antagonists have been shown. A more detailed localization of these two receptors in the basal ganglia is needed before such therapy can be tested in primates. Using electron microscopy immunohistochemistry, we found that A2AR is located mainly in striatopallidal GABAergic axon terminals in the external globus pallidus and in both pre- and postsynaptic structures in the striatum. We also found that A2AR was colocalized with mGluR5 in postsynaptic structures in the striatum. These data highlight numerous target sites whereby the combined use of A2AR and mGluR5 antagonists could mediate synergistic antiparkinsonian effects in primates. Finally, we studied the localization and electrophysiological effects of mGluR4, another non-dopaminergic target with good antiparkinsonian potential, in the monkey striatopallidal complex. We found that mGluR4 is located in both putative glutamatergic and GABAergic terminals and that activation of mGluR4 has multifarious effects on striatal and pallidal neurons, which showed increases and decreases in firing rates, as well as changes in firing patterns, in the striatopallidal complex of normal and MPTP-treated monkeys.
Together, these studies help to lay the foundation for the development of new non-dopaminergic antiparkinsonian treatment strategies in PD .
Table of Contents
Chapter 1:
Introduction…………………………………………………………..
1
1.1
Preface………………………………………………………………….
2
1.2
Rationale………………………………………………………………..
2
1.3 The striatum: Main entry to the basal ganglia
circuitry……………….. 5
1.3.1 Striatal medium spiny
neurons………………………………..
6
1.4 General basal ganglia
circuitry…………………………………………
7
1.5 Direct and indirect
pathways…………………………………………...
8
1.6 Basal ganglia
disorders…………………………………………………
11
1.6.1 Imbalanced activity of direct and indirect pathways in
Parkinson's
disease………………………………………………………………
13
1.6.2 Firing pattern abnormalities in Parkinson's
disease…………. 14
1.7 Animal models of Parkinson's
disease…………………………………
15
1.7.1 Acute and reversible models of
PD…………………………..
15
1.7.2 The 6-hydroxydopamine (OHDA)-treated rat model of Parkinson's
disease………………………………………………………………
16
1.7.3 The MPTP-treated monkey model of Parkinson's disease…..
17
1.7.3.1 The discovery of
MPTP…………………………….
18
1.7.3.2 Mechanism of action of
MPTP……………………..
19
1.8 Dopamine replacement
therapy………………………………………...
20
1.9 Non-dopaminergic antiparkinsonian drug
targets………………………
22
1.9.1 Cholinergic
receptors…………………………………………
23
1.9.2 Glutamate
receptors…………………………………………..
23
1.9.2.1 Ionotropic glutamate
receptors……………………..
24
1.9.2.2 Metabotropic glutamate
receptors…………………..
26
1.9.2.2.1
mGluR4…………………………………...
27
1.9.2.2.2
mGluR5…………………………………...
28
1.9.3 Adenosine A2A
receptors……………………………………...
30
1.9.4 mGluR5-D2R-A2AR
interactions……………………………..
32
1.10 Research
Summary……………………………………………………
34
1.10.1 Specific Aim
1………………………………………………
34
1.10.2 Specific Aim
2………………………………………………
35
1.10.3 Specific Aim
3………………………………………………
35
Chapter 2: Specific Aim 1: To examine the antiparkinsonian
effects of combined dopamine D2 receptor agonism/metabotropic
glutamate receptor 5 antagonism in MPTP-treated
monkeys…………………………………………………………...
37
2.1
Abstract…………………………………………………………………
38
2.2
Introduction………………………………………………….………….
39
2.3
Methods………………………………………………………………...
41
2.3.1
Animals……………………………………………………….
41
2.3.2 TH
immunolabeling…………………………………………..
41
2.3.3 Drug
administrations………………………………………….
43
2.3.4 Quantitative assessment of
parkinsonism…………………….
44
2.3.4.1 Quantification of
movements………………………
45
2.3.4.2 Counting of infrared beam
breaks…………………. 45
2.3.4.3 Parkinsonism rating
scale…………………………..
45
2.3.4.4 Eye blink
rate……………………………………….
46
2.4
Results………………………………………………………………......
48
2.4.1 TH
immunolabeling…………………………………………..
48
2.4.2 Quantification of
movements…………………………………
48
2.4.3 Infrared beam
breaks………………………………………….
49
2.4.4 Parkinsonism rating
scale…………………………………….
50
2.4.5 Eye blink
rate…………………………………………………
50
2.5
Discussion………………………………………………………………
51
2.5.1 mGluR5 antagonist as an antiparkinsonian
monotherapy…… 52
2.5.2 Potentiation of dopamine receptor-mediated antiparkinsonian
effects by mGluR5 antagonist: Differences between L-DOPA and
D2-like receptor
agonists……………………………………………………
53
2.5.3 Molecular interactions between mGluR5 and
D2LRs………. 54
2.5.4 mGluR5 antagonist/D2LR agonist combination as a potential
treatment for early
PD………………………………………………
55
Chapter 3: Specific Aim 2: To characterize adenosine A2A
receptor localization and co-localization with metabotropic
glutamate receptor 5 at the ultrastructural level in the monkey
basal
ganglia…………………………………………………………
56
3.1
Abstract…………………………………………………………………
57
3.2
Introduction……………………………………………………………..
58
3.3 Materials and
Methods………………………………………………….
60
3.3.1 Animals and tissue
preparation……………………………….
60
3.3.2 Antibody
characterization…………………………………….
61
3.3.3 Western
blots…………………………………………………
62
3.3.4 Single immunoperoxidase labeling for light
microscopy……. 64
3.3.5 Single immunoperoxidase labeling for electron
microscopy… 65
3.3.6 Single pre-embedding immunogold labeling for electron
microscopy………………………………………………………….
66
3.3.7 Dual pre-embedding immunogold/immunoperoxidase method for
striatal co-localization of A2AR and
mGluR5………………………
67
3.3.8 Analysis of
material…………………………………………..
68
3.3.8.1 Light microscopic
analysis…………………………
68
3.3.8.2 Electron microscopic
analysis………………………
68
3.4
Results…………………………………………………………………..
71
3.4.1 Western
blots…………………………………………………
71
3.4.2 Immunoperoxidase A2AR labeling: Light
microscopy………. 72
3.4.3 Ultrastructural localization of
A2AR………………………….
75
3.4.3.1 Immunoperoxidase A2AR
labeling…………………. 75
3.4.3.1.1
Putamen…………………………………...
78
3.4.3.1.2
GPe………………………………………..
78
3.4.3.1.3
SNr………………………………………..
80
3.4.3.2 Immunogold A2AR
labeling………………………...
80
3.4.3.3 A2AR/mGluR5
co-localization……………………...
81
3.5
Discussion………………………………………………………………
83
3.5.1 A2AR: A marker of indirect striatopallidal neurons in
primates and
non-primates………………………………………………………..
83
3.5.2 A2AR expression in striatal
terminals…………………………
86
3.5.3 A2AR expression in the
SNr………………………………….
87
3.5.4 A2AR in
glia………………………………………………….
88
3.5.5 Intracellular and extrasynaptic localization of A2AR in
the
striatum……………………………………………………………..
90
3.5.6 Postsynaptic co-cocalization of A2AR and mGluR5 in striatal
neurons: Potential sites of functional
interactions………………….
92
3.5.7 Therapeutic relevance of A2AR/mGluR5 antagonists in
Parkinson's
disease……………………………………………………………....
94
3.6
Acknowledgements……………………………………………………..
95
Chapter 4: Specific Aim 3: To characterize the subcellular
localization of metabotropic glutamate receptor 4 and examine the
electrophysiological effects of local infusion of metabotropic
glutamate receptor 4 agonists on firing rates and patterns of
neurons in the MPTP-treated monkey GPe and
putamen………... 97
4.1
Abstract…………………………………………………………………
98
4.2
Introduction…………………………………………………………….
99
4.3 Material and
methods…………………………………………………...
101
4.3.1
Animals……………………………………………………….
101
4.3.2
Immunohistochemistry……………………………………….
102
4.3.2.1 Pre-embedding immunoperoxidase for light
microscopy………………………………………………….
102
4.3.2.2 Pre-embedding immunoperoxidase for electron
microscopy………………………………………………….
104
4.3.2.3 EM observations and
analysis………………………
105
4.3.3
Electrophysiology…………………………………………….
105
4.3.3.1 Surgical
procedure………………………………….
106
4.3.3.2 Electrophysiological mapping of brain structures….
106
4.3.3.3 Intracerebral
injections……………………………...
108
4.3.3.4
Drugs………………………………………………..
109
4.3.3.5 Analysis of electrophysiological
data……………… 110
4.3.3.6 Characterization of striatal
neurons………………... 112
4.3.3.7 Data from normal
monkeys………………………...
113
4.4
Results…………………………………………………………………..
115
4.4.1 mGluR4 localization in the primate basal
ganglia…...………. 115
4.4.2 Ultrastructural analysis of mGluR4 in the
striatopallidal
complex………………………………….………………………….
117
4.4.3 Electrophysiological effects of group III mGluR
activation… 119
4.4.3.1 Basic firing characteristics of GPe neurons in normal vs.
MPTP-treated
monkeys…………………………………….
119
4.4.3.2 Group III mGluRs activation in the
GPe…………... 120
4.4.3.3 Effect of MPTP on basic firing characteristics of striatal
neurons……………………………………………………...
122
4.4.3.4 Group III mGluR activation in the
striatum……….. 123
4.5
Discussion………………………………………………………………
125
4.5.1 mGluR4 expression in the primate basal ganglia: Potential
significance towards mGluR4-mediated antiparkinsonian
effects… 126
4.5.2 mGluR4 in the striatum: A target for the regulation of
extrinsic glutamatergic and intrinsic GABAergic
circuitry…………………..
128
4.5.3 mGluR4 regulation of striatal and pallidal firing
rates……..... 129
4.5.4 Effects of group III mGluRs agonist vs. mGluR4 PAM on
striatal and pallidal
activity…………………………………………………
130
4.5.5 Concluding
remarks………………………………………......
131
4.6
Acknowledgements……………………………………………………..
132
Chapter 5:
Discussion……………………………………………………………..
133
5.1 Summary of findings and their implications for Parkinson's
disease…. 134
5.2 A critical look at
methodology…………………………………………
138
5.2.1
Immunohistochemistry……………………………………….
138
5.2.2
Electrophysiology…………………………………………….
140
5.2.3 Behavioral
experiments………………………………………
144
5.3 Future
directions………………………………………………………..
145
References………………………………………………………………………….
147
Non-printed
sources…………………………..…………………………….
194
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