Glutamatergic transmission is the main excitatory drive within the nervous system. Its effects are transmitted and controlled by many types of pre- and post-synaptic receptors segregated into two main categories based on sequence homology, structure, and signal transduction mechanisms. The ionotropic receptors, responsible for fast transmission, are ligand-gated ion channels. On the other hand, metabotropic receptors (mGluRs), responsible for slower, longer lasting effects, are G-protein-coupled receptors. The mGluRs are further subdivided into three groups (I, II, III) based on sequence homology, second messenger coupling and pharmacology. Availability of research tools, pharmacoagents, and an ability to selectively inhibit particular circuits has instigated an interest in group III mGluRs, because these receptors can be used as substrates for therapies alleviating symptoms in diseases exhibiting imbalances in glutamatergic transmission, such as Parkinson's disease. This study used high resolution electron microscopy immunohistochemical techniques to examine the localization and distribution of group III mGluRs in the striatum, which is the main input nucleus of the basal ganglia circuitry. In the striatum, group III mGluRs are shown to be localized mainly on presynaptic elements originating from both the cortico- and thalamo-striatal projections with significant degree of receptor co-expression in individual terminals. The group III mGluR-immunopositive terminals target, to a variable degree, postsynaptic elements of both the direct and indirect pathways, thus allowing for potential regulation of both striatofugal output systems. However, while the mGluR4-positive terminals seem to have a preference for the indirect pathway, the mGluR8-positive terminals are mainly associated with the direct pathway. On the other hand, the mGluR7-immunoreactive terminals are evenly distributed between the two output pathways. These data provide a map of the group III mGluRs localization along the main glutamatregic input pathways to the striatum, thereby offer a greater understanding of the mechanisms by which these receptors could be mediating their regulatory effects on excitatory transmission in the striatum and the basal ganglia. The anatomical schematic of receptor localization will further guide the discovery and development of pharmacoagents that can selectively activate group III mGluRs, regulate specific neuronal circuits , and hopefully address symptoms of numerous neurological disorders.
Table of Contents
TABLE OF CONTENTS 1. Introduction 1 1.1 Glutamate Receptors 2 1.1.1 Ionotropic Glutamate Receptor Classification 2 1.1.2 Metabotropic Glutamate Receptors Classification 5 1.1.3 Structure and Pharmacology of Group III mGluRs 6 22.214.171.124 Group III mGluR Structure and Composition 6 126.96.36.199 Group III mGluR Function and Physiology 8 1.1.4 Regional Localization of Group III mGluRs 15 188.8.131.52 Hippocampus 15 184.108.40.206 Retina 17 220.127.116.11 Cerebral Cortex 18 18.104.22.168 Thalamus 19 22.214.171.124 Basal Ganglia 20 1.1.5 Subcellular Localization of group III mGluRs 21 1.1.6 Electrophysiology of Group III mGluRs 24 1.2 Functional Circuitry of the Basal Ganglia 25 1.2.1 General Organization of the Basal Ganglia 25 1.2.2 Intrinsic Organization of the Striatum 28 1.2.3 Subtypes of Striatal Neurons 30 1.2.4 Corticostriatal Projections 31 1.2.5 Thalamostriatal Projections 32 1.2.6 vGluT expression in striatal afferents 33 1.2.7 Dopaminergic Projections 34 1.2.8 Dopamine Depletion and Parkinson's Disease pathophysiology 35 1.2.9 Glutamate Receptors and Parkinson's Disease Therapeutics 35 1.3 Study Objectives 37 1.3.1 Specific Aim 1 38 1.3.2 Specific Aim 2 39 2. Microcircuitry of mGluR4 and mGluR7 in the mouse striatum 40 2.1 Introduction 40 2.2 Materials and Methods 42 2.2.1 Animal and tissue preparation 42 2.2.2 Antibodies 42 2.2.3 Single Labeling Immunoperoxidase for Light Microscopy 45 2.2.4 Single and Double Pre-Embedding Immunoperoxidase for Electron Microscopy 46 2.2.5 Pre-embedding Immunoperoxidase and Immunogold for Electron Microscopy 47 2.2.6 Control experiments 48 2.2.7 Analysis of material 48 126.96.36.199 Immunoperoxidase material 48 188.8.131.52 Double-Immunoperoxidase 49 184.108.40.206 Immunogold material 49 220.127.116.11 Co-localization of mGluR8 with D1 using the double immunoperoxidase antibodies cocktail method or immunoperoxidase and immunogold 50 2.3 Results 51 2.3.1 Subcellular localization of Group III mGluRs in the mouse striatum 51 2.3.2 Group III mGluR colocalization in striatal glutamatergic terminals 53 2.3.3 Group III mGluR colocalization with vGluT1 and vGluT2 54 2.3.4 Group III mGluR innervation of striatal direct and indirect pathway neurons 58 2.4 Discussion 60 2.4.1 Technical considerations 62 2.4.2 Group III mGluRs are expressed in both corticostriatal and thalamostriatal glutamatergic afferents 64 2.4.3 Co-localization of Group III mGluRs in Glutamatergic Terminals 67 2.4.4 Group III mGluR innervations of direct and indirect pathways 68 2.4.5 Group III mGluRs in the Striatum-Potential Targets for Parkinson's disease 70 2.4.6 Concluding Remarks 71 3. Microcircuitry of metabotropic glutamate receptor 8 (mGluR8) in the mouse striatum 73 3.1 Introduction 73 3.2 Materials and Methods 76 3.2.1 Animal and tissue preparation 76 3.2.2 Antibodies 76 3.2.3 Immunoblotting 79 3.2.4 Single Labeling Immunoperoxidase for Light Microscopy 80 3.2.5 Single Pre-Embedding Immunoperoxidase for Electron Microscopy 81 3.2.6 Double immunoperoxidase labeling methods for mGluR8 and vGluT colocalization 82 3.2.7 Double immunoperoxidase labeling methods for mGluR8 and D1 83 3.2.8 Co-localization Studies: Pre-embedding Immunoperoxidase and Immunogold 84 3.2.9 Control experiments 85 3.2.10 Analysis of material 85 18.104.22.168 Overall distribution of mGluR8-labeled elements from single immunoperoxidase-stained material. 85 22.214.171.124 Co-localization of mGluR8 with vGluT1 or vGluT2 using the double immunoperoxidase antibodies cocktail method. 86 126.96.36.199 Co-localization of mGluR8 with D1 using the double immunoperoxidase antibodies cocktail method or immunoperoxidase and immunogold 86 3.3 Results 87 3.3.1 Specificity tests of mGluR8 Antibody 87 3.3.2 Quantitative Assessment of the Relative Abundance of Presynaptic mGluR8 Immunoreactivity in the Mouse Striatum 89 3.3.3 mGluR8 Innervation of Direct and Indirect Pathway Neurons 89 3.4 Discussion 92 3.4.1 Technical considerations 92 3.4.2 Physiological Effects of mGluR8 in Basal Ganglia 95 3.4.3 mGluR8-containing Glutamatergic Terminals Preferentially Target Direct Pathway Neurons 95 4. Conclusions and Future Directions 99 4.1 Group III mGluRs preferentially localize on presynaptic elements in mouse striatum 100 4.2 Co-localization of Group III mGluRs in Glutamatergic Terminals 102 4.3 Group III mGluRs have a differential preference for the D1-positive and D1-negative spines 105 4.4 Advantages & Technical Limitation 106 4.4.1 Light Microscopy 106 4.4.2 Pre-Embedding Immunoperoxidase 106 4.4.3 Pre-Embedding Immunogold 107 4.4.4 Double labeling using Pre-Embedding Immunoperoxidase and Immunogold 107 4.4.5 Concluding remarks 108 5. References and Citations 110 111 LIST OF TABLES 1.1 Group III mGluR mRNA and protein expression various CNS nuclei 28 LIST OF FIGURES 1.1 Downstream activation pathways following glutamate binding to group III mGluRs. 19 1.2 Diagram of the functional circuitry of basal ganglia in normal and Parkinsonian 34 2.1 mGluR4 and mGluR7 are mainly localized in presynaptic striatal elements 59 2.2 mGluR4 and mGluR7 display a significant degree of colocalization 62 2.3 Expression of mGluR4 and mGluR7 in vGluT-positive terminals 63 2.4 vGluT1- and vGluT2-immunoreactivity in the striatum. 66 2.5 Preferential targeting of D1-positive and D1-negative spines by mGluR-labeled terminals 68 3.1 mGluR8 immunoreactivity in wildtype and knockout mouse striatum 82 3.2 Preferential targeting of D1-positive and D1-negative spines by mGluR8-labeled terminals 91 4.1 Summary figure 103
About this thesis
|Committee Chair / Thesis Advisor|
|Microcircuitry of Group III Metabotropic Glutamate Receptors (mGluRs) in the Mouse Striatum ()||2018-08-28||