Synchronised bursting of the basal ganglia, specifically at frequencies below 30 Hz, has been implicated to have a major role in the pathophysiology of Parkinson's disease (PD). The aim of this thesis is to further investigate the pathological role of low frequency activities in the subthalamic nucleus. These activities are characterised through exploration of their interactions with oscillatory cortical activities as well as with subthalamic prokinetic high frequency activities and by determining their contribution to parkinsonian bradykinesia.

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The AS/AGU rat originated as a recessive mutation (agu) in a closed colony of Albino Swiss (AS) rats. The mutation is in the gene coding for the gamma isoform of protein kinase C. It is characterized by movement impairments and progressive dysfunction of the nigrostriatal dopaminergic (DA) and raphe striatal serotonergic (5-HT) systems. The movement impairments including rigidity of the hind limbs, a staggering gait, a tendency to fall over every few steps, a slight whole body tremor and difficulty in initiating movements. The dysfunction in both systems is characterised by a failure to release DA or 5-HT within the striatum and cell loss within the substantia nigra pars compacta (dopaminergic cells) and the dorsal raphe nuclei (5-HT+ve cells). In this study, three experiments were carried out to examine the possible pathological responses of midbrain cell groups to the agu mutation in the gene coding for protein kinase C-gamma (PKC-γ). Experiment 1 was carried out to examine levels of two groups of molecules in the midbrain cell groups using quantitative immunofluorescence microscopy of cell bodies or their surrounding neuropil (a) those molecules giving information about the capacity of midbrain aminergic cell bodies to synthesis transmitters; tyrosine hydroxylase (TH) in the dopaminergic neurons and serotonin (5-HT) in the serotonergic neurons (b) those which have been found to occur in human neurodegenerative conditions such as Parkinson’s disease: ubiquitin, parkin and α-synuclein (Lewy body proteins). Immunofluorescence levels of tyrosine hydroxylase (in dopaminergic cells of the SNC) and serotonin (in 5HT+ve cells of the dorsal raphe nuclei) were both significantly increased in AS/AGU (mutant) compared to the AS (control) rats aged 6 months and older. TH and 5-HT immunofluorescence levels were both significantly decreased in the striatum in the AS/AGU (mutant) compared to the AS (control) rat aged 12 months. Ubiquitin immunofluorescence show a gradual increase with age in AS and AS/AGU rats and the increase was much greater in the mutant in every region except the oculomotor and pontine nuclei. Parkin immunofluorescence show increases in the mutant within the SNC and the dorsal raphe nucleus and this increase was significant at older ages. Alpha-synuclein does not occur in the cell bodies of the substantia nigra or VTA but outside in the neuropil. Alpha-synuclein immunofluorescence levels progressively increased with age in both strains in the SN and VTA and were higher in the mutant. The levels of those molecules (ubiquitin, parkin and alpha-synuclein) do not differ in the striatum of mutants compared to controls. Experiment 2 examined SNC cell bodies to look for possible strain differences in cell size or ultrastructure or any sign of cell death using light and transmission electron microscopy. The diameter (maximum and minimum) of the SNC cells and nuclei were measured in toluidine blue paraffin wax and immunoperoxidase DAB staining for TH sections. Cell diameter was reduced in the AS/AGU mutant compared to the AS control. No obvious ultrastructural differences were seen in nigrostriatal neurons of both strains. The volume fractions of mitochondria and rough endoplasmic reticulum were significantly higher in the mutant. No Lewy bodies were present. Experiment 3 examined TH+ve nigrostriatal dopaminergic terminals in the dorsal caudate-putamen to determine whether there are (a) differences in the percentages and numbers of TH+ve terminals and (b) differences in synaptic vesicles numbers. In 12-month AS/AGU mutant, there are reduction in TH+ve terminals (40%) together with a reduction in vesicle numbers (40%) in such terminals where in 3-month AS/AGU mutant, the reduction in TH+ve terminals was more (50%) and a reduction in vesicles numbers by three quarters. TH-ve terminals are also reduced in numbers in 12 months aged AS/AGU mutant rats. In 12-month AS/AGU rats, there were significantly reduced numbers of synaptic terminals in the striatum compared to AS controls. This applied to both dopaminergic terminals (which make up 15% of the total) and to non-dopaminergic terminals. In 3-month AS/AGU rats, there is a reduction in terminal numbers, but this is restricted to the dopaminergic terminals only: non-dopaminergic ones are unaffected.

Background: This study aimed to evaluate the validity of the Edinburgh Depression Scale (EDS) as a screening measure for depression in patients with Parkinson’s disease (PD) and to compare the performance with the two questions recommended for depression screening by the National Institute for Health and Clinical Excellence (NICE) and the 15-item Geriatric Depression Scale (GDS-15). Methods: A sample of 120 patients attending PD out-patient clinics were interviewed in a standardised manner using the relevant sections of the Present State Examination-Schedules for Clinical Assessment in Neuropsychiatry to identify depression according to DSM-IV criteria. Participants then completed the three depression screening measures. Results: The sensitivity, specificity, positive and negative predictive values (PPV and NPV) of each screening measure for the identification of major and minor depression were calculated for different cut-off scores, and receiver operating characteristics (ROC) analyses were conducted. The EDS gave sensitivity of 74%, specificity of 92% (PPV 64%, NPV 95%) at the cut-off score of 10/11. The questions recommended by NICE gave sensitivity of 100% and specificity of 84% (PPV 54%, NPV 100%) at a threshold of 0/1, and the GDS-15 showed sensitivity of 84% and specificity of 89% (PPV 59%,NPV 97%) at a cut-off of 5/6. The areas under the ROC curve were 0.90, 0.95 and 0.92 respectively. The relative benefits of a two-stage screening process were explored. Conclusion: This study shows that the EDS, NICE questions and GDS-15 are valid screening measures for depression in PD provided the appropriate threshold scores are applied. The NICE questions showed the highest sensitivity but low PPV; a positive response to either of these two questions should highlight to the clinician that further diagnostic assessment of the patient may be warranted. A second screening measure, such as the EDS, could then be used to reduce the number of false-positives.

Parkinson's disease is an archetypal sporadic neurodegenerative disease: it is age-related and results in region-specific neurodegeneration with intracellular inclusions in many of the remaining neurones. Despite three decades of intense study, disease modifying therapies remain beyond the horizon, reflecting our understanding of the pathological process and the intrinsic challenges that the degenerative diseases of the brain present. Dysfunction in a number of cellular systems has been implicated in the development of Parkinson's disease. Protein homeostasis in terminally differentiated neurones is of particular import; unlike most other cells, they have no progeny with which to gift troublesome proteins. The ubiquitin proteasome system is the cell's main regulated proteolytic pathway and observations from neuropathological, genetic and animal studies have consistently found evidence that it may be aetiologically important in Parkinson's disease. Using a Cre/loxP approach Psmc1, which encodes an essential component of the 26S proteasome, was spatially deleted in catecholaminergic neurones in mice. This model reproduced both the region-specific neuronal loss and intraneuronal inclusions that define the sporadic human disease. In this work, I describe the further characterization of this unique model and use it to test hypotheses regarding the nature of the process that leads to these neuropathological features. Specifically, I show that in response to Psmcl deletion genes encoding sub- units of the 26S proteasome are up-regulated; using whole-genome microarray analysis I identify differentially expressed transcripts in micro dissected brain regions and then use the model to demonstrate that α-synuclein is not essential for the neurodegeneration or inclusion formation that result from Psmc1 deletion. These data underline the relevance of this model to the study of the pathogenesis of Parkinson's disease and identify avenues for further investigation.

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Synucleins are highly conserved presynaptic proteins with unknown function. α-synuclein plays a key role regulating dopamine homeostasis and is intimately involved in Parkinson’s disease (PD) pathogenesis. However, the normal/pathological role of α-synuclein remains unidentified. Studies exploring its function are limited as current transgenic mouse models do not fully recapitulate PD pathology. This thesis reports the functional characterisation of two novel synuclein-based mouse models. I report the molecular and functional characterisation of transgenic mouse lines with wild-type or A30P-mutant human α-synuclein genomic locus carried within a bacterial artificial chromosome. SNCA-A30P^+Snca-/- mice exhibited a highly physiologically relevant expression pattern of the transgene, including expression in the substantia nigra pars compacta (SNpc) and a specific, age-related loss of TH^+ cells in the SNpc, the key region of preferential cell loss in PD, compared with non-transgenic Snca -/- littermate controls. Analysis of dopamine signalling using fast-scan cyclic voltammetry (FCV) showed young adult SNCA-A30P^+Snca-/- mice had an approximately 20% lower evoked extracellular dopamine concentration ([DA]o) compared with non-transgenic Snca -/- littermate controls, a decrease specific to the dorsal striatum. This difference diminished with age and could not be attributed to changes in dopamine reuptake/content. I detail the behavioural and neurochemical phenotype in mice lacking all three synucleins (α/β/γ). Functional compensation between synucleins emphasises the importance of studying their effects by removing all three proteins simultaneously. Triple-null mice exhibited hyperactivity in a novel environment reminiscent of a hyperdopaminergic-like phenotype, but showed no phenotype in anxiety or motor related tests. FCV revealed synuclein triple-null mice had a two-fold increase in [DA]o, specific to the dorsal striatum and not attributable to changes in dopamine reuptake/content, changes in striatal nicotinic receptor activity nor calcium-dependent changes in dopamine exocytosis. Together, the analysis from these two novel mouse models reveal synucleins play an important role in altering synaptic function in the dorsal striatum (the region selectively affected in PD) and contributes to growing evidence suggesting synucleins are negative regulators of synaptic dopamine release.