This thesis focuses on sporadic parkinsonian syndromes that are associated with neurofibrillary degeneration and the accumulation of abnormal tau protein in the brain. The classic clinical presentation of corticobasal degeneration is a specific constellation of cortical and extrapyramidal signs, collectively termed corticobasal syndrome. The evaluation of all the archival cases with corticobasal degeneration in the Queen Square Brain Bank for Neurological Disorders reveals the high frequency of other phenotypic presentations. The result indicates that corticobasal degeneration commonly presents with a clinical picture, closely resembling progressive supranuclear palsy (PSP) or Richardson’s syndrome. On the other hand, cases with typical PSP pathology may occasionally present with a corticobasal syndrome. A quantitative assessment of the severity of tau pathology in different brain regions of the two phenotypic presentations of PSP reveals topographical differences that are closely linked with their respective clinical features. The features of repetitive finger tapping and handwriting in patients with PSP and Parkinson’s disease are compared and a distinct abnormality is identified in PSP which may be useful in differentiating PSP-parkinsonism from Parkinson’s disease. Twelve cases clinically presenting with a levodopa-responsive parkinsonian syndrome and post-mortem findings of nigral degeneration and predominant tau inclusions, which could not be readily classified into any recognised clinicopathological entity are also studied.

The aim of this study was to fully characterise the molecular and functional expression of TRPV channels in the retinal microvascular endothelium. Retinal microvascular endothelial cells (RMECs) were cultured from bovine retina. mRNA transcripts and protein expression was detected for TRPV1, 2, 3 and 4 using RT-PCR and western blots respectively, while fura-2-based Ca2+ microfluorimetry indicated TRPV channels were functional. Application of a TRPV1 agonist (resiniferatoxin; 10nM) to RMECs caused a transient rise in Ca2+. This was successfully abolished by pre-incubation with either of the TRPV1 antagonists capsazepine (5μM) or AMG9810 (1OμM). Ca2+ transients were also observed in response to 119-THC (1 OIJM) and were reduced after incubation with the TRPV2 inhibitor tranilast (75IJM). The TRPV3 agonist carvacrol (100pM) induced consistent calcium responses in RMECs while GSK1016790A (GSK; 100nM), a TRPV4 agonist, elicited transient rises in Ca2+ which were inhibited by HC067047 (1μM). Sub-cellular localisation of TRPV channels was examined in isolated RMECs and bovine retinal wholemounts using immunohistochemistry. Generally in RMECs, cytoplasmic staining for each TRPV channel was predominantly localised within a broad peri-nuclear zone. Interestingly, all TRPV channels, particularly TRPV3 and 4, were also detected in RMEC nuclei. Given the observed sub-cellular expression of TRPV4 in RMECs, the functionality of this channel was investigated further with confocal Ca2+ imaging. Application of GSK or 8'9 EET (an arachidonic acid metabolite) elicited transient Ca2+ signals in nuclear, mitochondrial and cytosolic regions of the cell. Inhibition of responses by HC067047 indicated TRPV4 activation was responsible and a possible involvement in EET-signalling. Low concentrations of GSK (1OμM) and 8'9 EET (1 nM) evoked isolated nuclear Ca2+ responses suggesting another role for TRPV4 in nuclear specific Ca2+ signalling. Detection of TRPV4 protein expression in nuclear protein extracts provided further evidence for such a role. Studies in this thesis have therefore reported functional TRPV expression, as well as highlighting potential roles for these channels, in RMECs.

This thesis focuses on understanding the nature of fixational eye gaze in humans. It tries to model eye gaze in a variety of methods: Descriptive statistics, wavelet analysis and it uses self similarity calculations to show that fixational eye-gaze indeed possess stochastic long range dependance and then it calculates Hurst parameter. This thesis also addresses the the practical side of eye-gaze. It develops a number of novel two- and three-dimensional applications to show the usefulness of eye gaze as a pointing device, in particular as it applied to patients with Locked-In syndrome. Among the applications is Daisy application which is a full featured word-processing software that shows the usefulness and ease of use of eye tracking as a pointing device. Furthermore, the thesis shows how eye tracking could be used in assisting surgeons during surgery by providing 3D navigation of a human organ.

Endothelial progenitor cells (EPCs) are vascular stem cells which hold the ability to repair and or replace damaged vasculature. EPCs have already been shown to improve patient outcomes in myocardial infarction and limb ischemia. Before EPCs can be considered for therapeutic angiogenesis in patients, we first must fully understand how these cells function and respond to the hypoxia milieu that is ever present in diabetic complications. This study has found chronic hypoxia affects OEC function and drives OECs towards a stress induced premature senescence. Chronic hypoxia markedly reduced OEC proliferation, downregulated markers commonly associated with stem and progenitor cells, reduced Akt and several of its substrates activation with increased SA-p-gal activity. The mRNA of members of the BMP/Smad pathway were increased in response to hypoxia however, this was not reflected at the protein level. OECs only responded to rhBMP-7 treatment with activation of the canonical Smad pathway. Gremlin a BMP antagonist which is also involved limb, lung and kidney development has recently been implicated in angiogenesis. This thesis further supports Gremlins role in angiogenesis as we report an increase in VEGFR2 activation which enhanced OEC angiogenic potential through increased tube formation and migration in OECs with rhGremlin treatment. Since Gremlin is known to be essential in the limb, lung and kidney formation its potential involvement in eye and retinal vasculature development was determined through the generation of grem1-/- mice. Our data suggests Gremlin may not be critical in the eye and retina development as the eyes of grem1-/- mice were comparable to the wild type control mice. However, there was a slight decrease in the number of branching retinal vessels which suggests Gremlin may regulate the vascularisation of the retina. In summary, data in this thesis advances our understanding of OEC function under hypoxic conditions and highlights novel roles for BMP-7 and Gremlin in OEC biology and blood vessel formation.

Cerebral perfusion, or cerebral blood flow, CBF, is a physiological variable that measures the amount of blood delivered to brain tissue per unit time. In neonates, CBF assumes an important role as alterations in CBF are linked to many instances of brain injury, particularly following preterm or complicated births. CBF can be measured using a number of techniques, most of which rely on ionising radiation and require the administration of exogenous substances. This renders them unsuitable for studying CBF in healthy neonates and for repeated studies. In this thesis, Magnetic Resonance Imaging (MRI) techniques capable of measuring CBF non-invasively are optimised for the neonatal population. The longitudinal relaxation time constant, T1, of blood is an important parameter in many MRI techniques, such as perfusion quantification using Arterial Spin Labelling (ASL). In most applications, ex-vivo literature values are used for blood T1. A novel method to measure blood T1 in vivo in a very short time is presented. It is found that blood T1 values in neonates are very variable and strongly correlated to the haematocrit. This method can be used to improve CBF quantification using ASL in neonates. A robust method used to measure mean CBF is also presented. Firstly, the mean flow to the brain in the arteries of the neck was measured using an optimised phase-contrast angiography protocol. This was then divided by the brain volume, computed from an anatomical MR image, to yield mean CBF. This method was applied to a small cohort of infants, where the relationship between CBF and postmenstrual age was investigated. Arterial Spin Labelling was also used in both adults and neonates. In adults, CBF values were compared to those obtained using the PCA method. Techniques to estimate the parameters needed in the ASL model for CBF quantification were also explored. Finally, ASL data acquired in neonates is presented and further improvements to CBF measurements using ASL in this age group are discussed.

Recent findings have demonstrated fibroblast growth factor-20 (FGF20) to have neuroprotective effects on dopamine neurones in vitro. In this thesis, FGF20’s neuroprotective effects on dopamine neurones were further investigated. A ventral mesencephalic (VM) embryonic dopamine neurone culture system and a partially lesioned 6-hydroxydopamine (6OHDA) rat model of Parkinson’s disease (PD) were established in which FGF20 was evaluated for its neuroprotective effects both in vitro and in vivo. Using immunohistochemistry, FGF20 and at least three of its receptors (fibroblast growth factor receptor (FGFR) 1,3, and 4) were demonstrated to be localised to dopamine neurones and glial cells in the rat nigrostriatal tract and in VM embryonic dopamine neurone cultures. In vitro, FGF20 protected VM embryonic dopamine neurones against 6OHDA toxicity, and, in vivo, chronic supra-nigral delivery of FGF20 protected nigrostriatal dopamine neurones against a partial 6OHDA lesion. Importantly, FGF20 also preserved motor function in the 6OHDA lesioned rats. In a separate in vivo study, experiments were carried out to investigate whether pharmacological inhibition of FGFR activation is able to potentiate 6OHDA-induced nigrostriatal degeneration in the rat, and results from this study suggest that the endogenous FGF system might play a protective role in the nigrostriatal tract. Additionally, in PC 12 cells, FGF20’s neuroprotective effects against 6OHDA toxicity were demonstrated to be mediated by the FGFRs at the receptor level, and by the ERK1/2 MAPK pathway at the intracellular level. Others have shown the heparin sulphate proteoglycan, agrin to potentiate FGF2 stimulated ERK1/2 activation and neurite outgrowth in PC12 cells. It was demonstrated here that agrin potentiates FGF20 stimulated ERK1/2 activation, but it fails to potentiate FGF20’s neuroprotective effects in PC12 cells.

The dLGN (dorsal Lateral Geniculate Nucleus) is the gateway to cortical processing for visual information encoded by RGCs (retinal ganglion cells). Owing to advances in genetic manipulations, the mouse is increasingly the model system of choice for understanding the function and organisation of the visual system. However the mouse dLGN has not been thoroughly investigated anatomically due to its difficult topology and location in the brain. We addressed this issue by indentifying the best reconstruction paradigm for recovering the true 3D shape of the dLGN from serial histological sections. We have placed each animal into standard space coordinates which allows for analysis on a population level rather than on an individual basis. Having investigated several different strategies, the optimal process proved to be the manual alignment of individual sections to a MRI template (Align3TP program) followed by an algorithm that corrects for the local deformations of individual sections (RegFSD program). Our anatomical investigations into the geniculo-cortical projection used discrete cortical injections of fluorescent microspheres (RetroBeads). This has allowed us to quantitatively define projection columns in 3D, and investigate how they vary with the location of the cortical injection. Furthermore, the orientation, spread, and cellular components of the column were investigated to search for defining characteristics of projection columns, using PCA (principal component analysis). Our population study in standard space has allowed us to pool data across animals and investigate the topographic order of the geniculo-cortical projection. We have investigated different metrics for examining the precision of the geniculo-cortical projection in 3D. The data shows how altered neonatal spontaneous activity in the AChRnp2~~ mutant affects the geniculo-cortical projection column.

Neurons of the adult mammalian central nervous system (CNS) and peripheral nervous system (PNS) differ in their response to injury. PNS neurons have a robust growth response and are capable of regenerating their axons and reinnervating peripheral targets following nerve injury. However, despite this robust regenerative response, specificity of target reinnervation is often poor, leading to a suboptimal functional outcome. In contrast, injured CNS neurons have a poor growth response and completely fail to regenerate, but uninjured axons that survive the injury undergo some degree of spontaneous reorganisation that is accompanied by limited functional improvement. In theory, appropriate reorganisation of central connections could compensate for topographical errors in the periphery and enhancing the innate capacity for reorganisation after CNS injury could compensate for lost function. Thus, recovery from both types of injury may benefit from strategies to enhance CNS plasticity. This Thesis makes use of an experimental strategy to promote plastic changes, that is, the enzyme chondroitinase ABC (ChABC) which degrades chondroitin sulphate proteoglycans - a major class of inhibitory extracellular matrix molecules. Viral vector technology was used to optimise the delivery of ChABC by gene transfer to host spinal cord cells. A lentiviral vector expressing the ChABC gene (LV-ChABC) was delivered to the adult rat spinal cord to achieve long-term delivery of active ChABC in vivo. After peripheral nerve injury, where two forelimb nerves were cut and repaired with different levels of inaccuracy, electrophysiological studies demonstrated that LV-ChABC injection led to significant plasticity of central connections, such as reorganisation of high and low threshold polysynaptic reflexes. ChABC did not lead to any changes in reflex activity in uninjured animals. These results indicate that ChABC facilitates the amplification of compensatory changes in the spinal cord following injury to the periphery. ChABC treatment was also investigated in the context of CNS injury. Following contusion injury, a clinically relevant model which mimics the functional and pathological characteristics typical of a human spinal cord injury, LV-ChABC injection led to significant functional improvements, including enhanced conduction, reorganisation of spinal reflexes below the lesion and improved performance on a sensorimotor behavioural task. in vivo. as Finally, viral vector technology was used to develop a novel tool for the study of anatomical plasticity. By expressing the construct synaptopHluorin (SpH), lentiviral and adenoviral-associated viral vectors were used to label presynaptic terminals in vitro and in vivo. As the pHluorin component of the label is pH sensitive, this tool has the potential additional advantage of enabling the visualisation of synaptic function The SpH vector was used to label a major motor pathway, the corticospinal tract, well as sensory neurons, and to visualise their terminals as SpH-labelled puncta. Using this tool, it will be possible to study synaptogenesis in a quantifiable manner, with the potential to study the activity of these new synaptic terminals. Thus, this Thesis uses a gene delivery approach to deliver ChABC to the spinal cord after central or peripheral neuronal injury, leading to the promotion of plasticity and functional repair. The development of a novel tool for visualising synapses is also described, presenting a novel opportunity for the study of plasticity after neuronal injury.

Rodents have a topographic map in primary somatosensory cortex of the contralateral facial whiskers. A brief period of whisker trimming causes the representation of the nontrimmed whiskers (spared) to expand into the cortex that has lost its principal sensory input (deprived). It has been hypothesized that this is mediated by a period of persistent disinhibition in deprived cortex that enables the expansion of spared whisker representations. Alternatively, it has been proposed that inhibition undergoes a biphasic change with an initial, brief period of disinhibition to promote plasticity in excitatory circuits followed by a more prolonged increase in inhibition to re-establish the excitatory – inhibitory balance. These hypotheses make different predictions about how inhibition changes during cortical map plasticity, which I have tested in this thesis. I focused on fast-spiking (FS) interneurons, which are thought to play an important role in adult cortical plasticity. I made electrophysiological recordings in layer 2/3 to determine how inhibitory circuitry in deprived cortex is affected by whisker deprivation. The amplitude of miniature excitatory postsynaptic potentials (mEPSPs) in deprived FS interneurons was increased with no change in mEPSP frequency suggesting that the global excitatory drive onto FS interneurons was potentiated. In contrast, the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) in layer 2/3 pyramidal neurons was unchanged, but there was a small but significant increase in mIPSC frequency. I investigated feedback inhibitory circuits in more detail by recording from pairs of pyramidal cells and FS interneurons that were synaptically-connected. Surprisingly, I found that the strength of local excitation onto FS interneurons and the strength of FS – mediated inhibition on deprived pyramidal neurons were unchanged. I concluded that, contrary to two popular hypotheses, a brief period of sensory deprivation did not alter the feedback inhibition in layer 2/3 of deprived cortex.

The studies described in this thesis explore the molecular mechanisms by which nutrition modulates Adult Hippocamapal Neurogenesis (AHN) using a human hippocampal progenitor cell line (HPC). AHN has been shown to be important for cognition and mood regulation. Interestingly, diet in the form of the Omega-3 fatty acids Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DMA) is known to have beneficial effects on cognition and mood; these effects are hypothesised to be mediated via modulating AHN. My studies show that in an in vitro model of stress, EPA (10uM) and DHA (10uM) prevent the detrimental effects of Cortisol on proliferation and neurogenesis by increasing the percentage of dividing cells and neurogenesis while decreasing apoptosis mainly by promoting survival. Resveratrol (1uM), a stilbenoid present in the skin of red fruit, prevents the Cortisol induced changes by increasing the percentage of dividing cells and neurogenesis but has no effect on apoptosis. Intermittent fasting (IF), shown to promote AHN, increases the expression of Klotho, the longevity gene. Klotho over expression increases neurogenesis. Whereas Klotho knock down decreases neurogenesis by diminishing survival. To further investigate the molecular pathways downstream of Resveratrol as mimetic of IF and Klotho expression, I focused on PPARy (Peroxisome proliferator-activated receptor) a nuclear receptor transcription factor which is known to activate Klotho transcription and is activated by Resveratrol. My results show that Resveratrol partly requires PPARy to activate Klotho and that Klotho is partly required during proliferation for Resveratrol to exert its effect. Whereas, Resveratrol partly requires both Klotho and PPARy to increase neurogenesis and PPARy requires Klotho to increase the proportion of mature neurons. These experiments provide evidence that PPARy and Klotho are two of the neurogenic effectors of Resveratrol. This thesis provides for the first time evidence in a human in vitro model of neurogenesis and stress that EPA, DHA and Resveratrol can modulate neurogenesis and prevent its decrease induced by stress. This study also provides the first identification of Klotho and PPARy as downstream effectors of Resveratrol on neurogenesis.