Comparative Analysis of Cortical Innervation Across Functional Regions of the Subthalamic Nucleus in Healthy and Parkinsonian Monkeys Open Access

Abstract

Background: Parkinson’s disease (PD) is characterized by motor deficits but also involves non-motor symptoms, including apathy and cognitive deficits. Previous studies in parkinsonian animals have shown that PD leads to decreased cortical innervation of the sensorimotor region of the subthalamic nucleus (STN), resulting in dysfunction of information transmission along the cortico-subthalamo-pallidal pathway. Whether such cortical denervation also affects the associative and limbic regions of the STN remains unclear.

Methods: To assess changes in STN cortical innervation, we quantified the number and total volume of cortical vGluT1 terminals in the limbic, associative and sensorimotor regions of the STN using confocal microscopy. To evaluate potential changes in the pattern of synaptic connectivity of cortico-subthalamic terminals in parkinsonian animals, transmission electron microscopy was used.

Results: Confocal analyses revealed a significant reduction in the total volume, but no alteration in the density, of vGluT1-positive puncta in the STN. EM analyses revealed no differences in terminal surface area between conditions, however terminals in the limbic region of the STN were significantly smaller than other regions regardless of condition. Notably, we identified axo-axonic contacts formed by vGluT1 terminals, a synaptic arrangement not previously reported in the macaque or rat STN but occasionally seen in cat.

Conclusion: Our findings demonstrate that dopamine depletion in parkinsonism is associated with structural remodeling of cortico-subthalamic glutamatergic terminals. Although the number of vGluT1- positive puncta was preserved, the total volume they occupy within the STN was reduced, suggesting a shrinkage of the space that the puncta occupy. Additionally, our EM data show that the limbic region contains smaller vGluT1 terminals (surface area) than the associative and sensorimotor region regardless of condition. At the ultrastructural level, we identified axo-axonic synapses formed by vGluT1-positive terminals, a synaptic relationship that has not been described in the primate or rodent STN. Taken together, these results support the hypothesis that cortico-subthalamic inputs undergo plasticity in the parkinsonian state, likely contributing to impaired cortical control of STN activity and altered basal ganglia network dynamics. 

Table of Contents

Table of Contents

List of Tables ....................................................................................................................................................................... 11

List of Figures ...................................................................................................................................................................... 11

Chapter 1: Introduction ...........................................................................................................................................................1

1.0 Basal ganglia and functional connectivity ............................................................................................................2

1.1 Parkinson’s Disease ........................................................................................................................................2 1.2 Functional and connectional alterations of basal ganglia thalamocortical circuits in parkinsonism

circuits 3

1.3 Functional regions of the STN..............................................................................................................................4

1.4 Limbic and cognitive dysfunctions in PD ...........................................................................................................5

1.5 Changes in Cortical connectivity in the STN......................................................................................................5

1.6 Aims of this study....................................................................................................................................................6

Chapter 2: Methods & Statistics.............................................................................................................................................9

2.0 Animals and Induction of parkinsonism........................................................................................................... 10

2.1 Animal euthanasia, tissue fixation and processing, and analysis of dopaminergic denervation. .............. 10

2.2 Immunofluorescence staining of vGluT1 for confocal imaging................................................................... 11

2.2 Confocal microscopy ........................................................................................................................................... 12

2.4 Imaris: 3D surface reconstruction...................................................................................................................... 15

2.5 Statistical Analysis of vGluT1- immunostained puncta at the confocal microscopic level. ..................... 16

2.6 Immunoperoxidase staining of vGluT1 for EM imaging. ............................................................................. 16

2.7 Electron microscopy data collection ................................................................................................................. 17

2.8 Analysis of vGluT1-positive terminal surface area and categorization of post synaptic targets .............. 18

2.9 Statistical analysis of vGluT1-immunostained elements at the EM level .................................................... 18

Chapter 3: Results of Confocal Microscopy Studies ................................................................................................................ 22

3.0 vGluT1 terminal-like puncta density in the STN of parkinsonian and control monkeys......................... 23

3.1 Comparison of vGluT1 terminal-like puncta density in different functional regions of the STN

between parkinsonian and control monkeys .......................................................................................................... 23

3.2 Volume distribution of vGluT1-positive terminals in STN functional regions of control and

parkinsonian monkeys ................................................................................................................................................ 24

Chapter 4: Electron Microscopy Results ................................................................................................................................ 30

4.0 vGluT1 terminal surface area in the STN of parkinsonian and control monkeys ..................................... 31

4.1 Post-synaptic targets of vGluT1 terminals in the STN of parkinsonian and control monkeys ............... 31

Chapter 5: Discussion........................................................................................................................................................... 37

5.0 Discussion.............................................................................................................................................................. 38

5.1 Variability in Cortico-Subthalamic Innervation Across Studies and Parkinsonian Monkeys................... 38

5.2 Ultrastructural characterization of vGluT1 terminals in the STN ................................................................ 40

5.3 Implications of plastic changes in cortico-STN projections in parkinsonism ............................................ 42

5.4 Future directions................................................................................................................................................... 42

5.5 Limitations ............................................................................................................................................................. 43

Chapter 6: Supplementary Materials..................................................................................................................................... 45

Supplementary Table 1. EM data ............................................................................................................................. 46

Supplementary Table 2. Characteristics of animals used in Mathai et al. 2015 ................................................. 47

Supplementary Figure 1. MR257R parkinsonian distributions of vGlut1 elements in confocal images....... 48

Supplementary Figure 2. MR273R parkinsonian distributions of vGlut1 elements in confocal images....... 49

Supplementary Figure 3. MR400L parkinsonian distributions of vGlut1 elements in confocal images ....... 50

Supplementary Figure 4. MR317R control distributions of vGlut1 elements in confocal images................. 51

Supplementary Figure 5. MR359R control distributions of vGlut1 elements in confocal images................. 52

Supplementary Figure 6. MR380L control distributions of vGlut1 elements in confocal images ................. 53

Supplementary Figure 7. MR273R parkinsonian distribution plots EM data. .................................................. 54

Supplementary Figure 8. MR321L parkinsonian distribution plots EM data.................................................... 55

Supplementary Figure 9. MR400L parkinsonian distribution plots EM data.................................................... 56

Supplementary Figure 10. MR317R control distribution plots EM data. .......................................................... 57

Supplementary Figure 11. MR359R control distribution plots EM data. .......................................................... 58

Supplementary Figure 12. MR380L control distribution plots EM data............................................................ 59 

About this Master's Thesis

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Biology, Histology Biology, Neuroscience
Keyword	vGluT1 Parkinsonian rhesus macaques Parkinson's Disease Cortico-subthalamic projection Subthalamic Nucleus
Committee Chair / Thesis Advisor	Adriana Galvan, Emory University Yoland Smith, Emory University
Committee Members	Jessica Raper, Emory University Thomas Wichmann, Emory University

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