Metabotropic glutamate and adenosine receptors in the monkey striatopallidal complex and their involvement in Parkinsonism: Anatomy, physiology, and behavior Open Access

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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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	parkinson's disease glutamate adenosine metabotropic monkey
Committee Chair / Thesis Advisor	Smith, Yoland, Emory University
Committee Members	Wichmann, Thomas, Emory University Hall, Randy A, Emory University Muly, E Christopher, Emory University Factor, Stewart, Emory University

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