Glutamatergic Inputs to the Monkey Subthalamic Nucleus: A Comparison of Relative Abundance, Synaptology and Functional Connectivity in Normal versus Parkinsonian Conditions. Open Access

Mathai, Abraham (2013)

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Abnormal activity in the subthalamic nucleus (STN) has been linked to motor and non-motor abnormalities in Parkinson's disease. In addition to abundant GABAergic inputs from the external globus pallidus, the STN also receives significant glutamatergic afferents from the cerebral cortex, thalamus and brainstem. Although the sources of these excitatory afferents have been recognized, their pattern of synaptic connectivity and relative prevalence in normal and pathological conditions are unknown. Hence, we undertook an ultrastructural analysis of the abundance and synaptology of glutamatergic terminals in the STN of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treated parkinsonian monkeys, using vesicular glutamate transporters 1 and 2 as markers of cortical (vGluT1) or sub-cortical (vGluT2) glutamatergic inputs. Because the STN is functionally divided into a "motor" sector and a "non-motor" territory, that receive inputs from different regions, the distribution and density of vGluT1- and vGluT2-positive terminals were compared between these two regions in normal and parkinsonian conditions.

In the normal STN, vGluT1-positive terminals (~14,000/mm2) were more abundant than vGluT2-positive terminals (~10,300/mm2). vGluT1-immunoreactive terminals innervated dendritic spines (~30%) and dendritic shafts (~70%); whereas, vGluT2-immunoreactive terminals almost exclusively targeted dendritic shafts (~90%). The dendritic shaft innervation sites of both glutamatergic inputs were mostly situated on the distal parts of STN dendrites. Notably, we found that the relative density of both vGluT1- and vGluT2-positive terminals significantly decreased by almost half in parkinsonian monkeys compared with controls. Still, the innervation patterns of both vGluT1- and vGluT2-immunopositive inputs on STN dendritic trees were quite similar between the normal and parkinsonian states. No major differences were observed in the relative abundance and synaptology of these glutamatergic inputs between the motor and non-motor STN.

Furthermore, preliminary electrophysiology data from 2 monkeys showed that fewer pallidal neurons responded to electrical stimulation of the internal capsule with the characteristic early excitation (<12ms) typically mediated by corticosubthalamic activation. These data suggest that partial degeneration of cortical inputs to the STN may affect transmission along the cortico-subthalamo-pallidal network in parkinsonism.

Whether the loss of glutamatergic inputs to the STN in parkinsonism is a primary pathological phenomenon or a compensatory mechanism is unclear.

Table of Contents

1. Introduction 1
1.1. Functional Circuitry of the Basal Ganglia 1
1.1.1. General Organization of the basal ganglia 1
1.1.2. Basal ganglia circuits 2
1.1.3. Parallel processing in the basal ganglia 3
1.1.4. Regulation of basal ganglia function by dopamine: Implications in pathological conditions 6
1.2. Organization of the STN 6
1.2.1. Functional topography of the STN 7
1.2.2. Morphology of STN neurons 9
1.2.3. Glutamatergic afferents to the STN 10
1.3. Pathophysiology of the STN in Parkinson's disease 16
1.4. STN deep brain stimulation (DBS) as a treatment for Parkinson's disease 19
1.5. Specific Aims 20
1.5.1. Specific Aim 1 21
1.5.2. Specific Aim 2 22

2. Cortical Innervation of the Subthalamic Nucleus Decreases in Experimental Parkinsonism 23
2.1. Introduction 23
2.2. Methods 25
2.2.1. Animals 25
2.2.2. Induction of Parkinsonism 26
2.2.3. Animal euthanasia and tissue fixation 27
2.2.4. Anatomical Experiments 27
2.2.5. TH immunostaining 35
2.2.6. Electrophysiological Experiments 35
2.3. Results 39
2.3.1. State of Parkinsonian Motor Symptoms and Nigrostriatal Dopaminergic Pathology in MPTP-treated Monkeys 39
2.3.2. Lack of vGluT1 and vGluT2 co-localization in the Monkey STN 42
2.3.3. Changes in vGluT1-immunopositive innervation of the dorsolateral STN in MPTP-treated monkeys 45
2.3.4. The pattern of innervation of STN neurons by vGluT1-positive terminals is unchanged between normal and parkinsonian monkeys 52
2.3.5. Physiological impact of corticosubthalamic activation on pallidal neurons between normal and parkinsonian monkeys 56
2.4. Discussion 60

3. Loss of Motor and Non-motor Glutamatergic Inputs to the Subthalamic Nucleus in MPTP-treated parkinsonian monkeys 68
3.1. Introduction 68
3.2. Methods 70
3.2.1. Animals 70
3.2.2. Induction of Parkinsonism 70
3.2.3. Animal euthanasia and tissue fixation 70
3.2.4. Tissue processing 71
3.2.5. STN volume measurements 71
3.2.6. Double Immuno EM for vGluT1 and vGluT2 71
3.2.7. Analysis of EM material 74
3.2.8. TH immunostaining 75
3.3. Results 75
3.3.1. vGluT1 and vGluT2 co-localization in the monkey STN 75
3.3.2. Relative abundance of vGluT1- and vGluT2-containing terminals in the 'motor' and 'non-motor' territories of the STN 76
3.3.3. Dendritic innervation patterns of vGluT1- and vGluT2-containing terminals in the monkey STN 79
3.3.4. Relative abundance and synaptology of vGluT1- and vGluT2-containing terminals in normal versus parkinsonian monkeys 85
3.4. Discussion 93

4. Conclusions and Future Directions 99
4.1. Conclusions 99
4.1.1. Both the motor and non-motor territories of the STN receive significant cortical and sub-cortical glutamatergic afferents 99
4.1.2. Cortical and sub-cortical glutamatergic inputs innervate different parts of the dendritic tree of STN neurons 101
4.1.3. Both cortical and sub-cortical glutamatergic inputs to the STN are partially lost in MPTP-treated parkinsonian monkeys 104
4.1.4. Potential changes in the functional impact of the hyperdirect corticosubthalamic pathway upon pallidal neurons in parkinsonism 106
4.2. Future Directions 110
4.2.1. Role of the STN in non-motor functions of the basal ganglia 110
4.2.2. Integration of functionally distinct information in the STN 111
4.2.3. Influences of cortical versus sub-cortical glutamatergic afferents on activity of STN neurons 112
4.2.4. Functions of the STN in basal ganglia mediated action selection programs 112
4.2.5. Do glutamatergic inputs to the striatum and STN arise from single neurons within the cortex and/or thalamus in primates? 113
4.2.6. Functional changes to corticosubthalamic and subcorticosubthalamic excitatory inputs in parkinsonism 113
4.2.7. Plastic changes in response to the functional loss of glutamatergic inputs to the STN: Quantification of glutamate receptors on cortical and sub-cortical glutamatergic afferents to the STN 115
4.2.8. Common pathological mechanisms affecting the integrity of glutamatergic afferents to the striatum and subthalamic nucleus in the dopamine-denervated state 116
4.2.9. Impact of the loss of corticosubthalamic terminals on the efficacy of STN-DBS 117
4.3. Concluding Remarks 117

5. References 119

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