The main aim of this thesis was to examine the role of metabotropic glutamate receptors (mGluRs) in the control of the excitability of sympathetic preganglionic neurones (SPNs). A review of the literature concerning SPNs and glutamate receptors has been performed. Experiments were made using whole-cell patch-clamp recordings from neurone in slices of spinal cord. During recording neurones were perfused with either Lucifer yellow or biocytin and following recording the slices were processed and the morphology of the neurones examined. SPNs were identified by their characteristic electrophysiological and morphological properties. The pharmacology of mGluR agonist-induced changes in the excitability of SPNs was examined. Almost all SPNs demonstrated a depolarisation with a pharmacological profile suggesting that it was mediated by a group I mGluR. A minority of SPNs also demonstrated hyperpolarising responses to agonists with activity at group II mGluRs, suggesting that activation of group II mGluRs may have inhibitory effects on some SPNs. Activation of mGluRs modulated the frequency of ongoing membrane potential oscillations and induced membrane potential oscillations in a subpopulation of previously silent SPNs. Responses involving induction of oscillations or increases in the frequency of spontaneous oscillations had agoinst profiles suggesting involvement of a group I mGluR, and responses involving decreases in the frequency of oscillations had agonists profiles suggesting that they were mediated by a group II mGluR. This study has demonstrated excitatory and inhibitory effects mediated by activation of different mGluRs on SPNs and has investigated some of the underlying mechanisms. Activation of mGluRs was also shown to modulate glutamatergic synaptic inputs to SPNs. Synaptically evoked responses mediated by mGluRs remain to be demonstrated.

Introduction: Ependymoma is the second most common malignant brain tumour of childhood. 50% of children with primary disease recur; three-quarters of these do not achieve long term survival. In the ‘molecular era’ of cancer research, diagnosis combines advanced molecular profiling with histopathological assessment. Whilst primary ependymomas can be classified based on epigenetic and transcriptomic features, there is little information on molecular signatures at recurrence. However, some small studies have implicated cancer immunity. Trials of novel therapies at recurrence have been disappointing. This study undertook molecular profiling of recurrent ependymoma, combined with contemporary clinical data, to better understand recurrence biology and potential therapy options. Methods: Clinical outcomes for 188 children with recurrent ependymoma were analysed. Cases with primary and matched recurrent samples were included in DNA methylation (n=56), RNA sequencing (n=52) and immunohistochemical (IHC) (n=56) analyses. RNA sequencing from FFPE tissue was validated to expand the cohort. Results: Recurrence was the strongest predictor of long term survival. Treatment approach at primary diagnosis was not associated with survival, but radiotherapy at first recurrence was associated with better short-term outcomes. Children with the commonest DNA methylation based diagnoses, EPN_PFA and EPN_RELA, had equally poor outcomes. RNA sequencing from FFPE tissue was effective, therefore tumours sequenced from FFPE and FF tissue were included in paired gene expression analyses. Transcriptomic and DNA methylation analyses identified three similar subgroups in FFPE and FF cohorts (PF1, PF2 and ST). At first recurrence, PF1 was associated with downregulated immune and inflammatory ontologies, which may indicate tumour immune escape. PF2 and ST subgroups demonstrated upregulation of ontologies associated with adaptive immunity. Despite this, there was little evidence of change in either immunogenicity or T-cell effector activity at first recurrence. IHC analysis identified a fall in inflammatory cells in posterior fossa tumours at recurrence and indicated that ependymoma is an immune excluded tumour. Conclusions: This study highlights both the abysmal prognosis for this disease, and the need for a better understanding of tumour biology to improve outcomes. This study has contributed novel data on changes at recurrence across molecular subgroups, and identified the immune excluded nature of ependymoma, which may be important in guiding therapy. The validation of RNA-seq from FFPE in childhood brain tumours has facilitated access to a large set of previously uninvestigated samples.

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Introduction: Carotid artery stenosis is associated with stroke and large randomised controlled trials (RCTs) have shown that carotid endarterectomy (CEA) can reduce subsequent stroke risk in those with symptomatic 50-99% carotid stenosis. However, not all patients benefit from CEA equally and therefore other methods of risk stratification are needed. Non-invasive plaque imaging with ultrasound (US) and Magnetic Resonance Imaging (MRI) have shown potential in this regard, but ultrasound is much cheaper and more widely available. Both are associated with plaque instability features on histology and relate to symptom status. There a number of studies in asymptomatic patient cohorts, but only one that shows echolucent plaques on US are a risk predictor for stroke and TIA. Further, there is limited longitudinal data on the predictive value of plaque echolucency or other US features in recently symptomatic patients. We have previously shown that the presence of hyperintense signal in the carotid artery on MRI relates to plaque haemorrhage (MIR-PH) and is a strong predictor of recurrent stroke. However, the temporal stability of the MIR-PH signal is not known for beyond 12 months and how it is linked stenosis progression. The aims of this thesis are to investigate US features for carotid plaque instability and if they can predict stroke in a cohort study of symptomatic patients. These were also compared to MRI carotid plaque imaging and a clinical risk score. Methods: Patients mild to moderate carotid stenosis, not planned for CEA, were recruited into a prospective cohort study. They had ultrasound plaque imaging was performed and five features were compared to risk markers for stroke in a cross-sectional design. The presence of plaque haemorrhage on MRI (MRI-PH) was determined and the Carotid Artery Risk (CAR) score calculated. Patients were followed up until the end of the study or event. In another cohort of patients, serial MRI and US scans were performed to determine the temporal stability of the MRI-PH signal and its effect on stenosis progression. Results: In a cross-sectional cohort study (91 symptomatic and 85 asymptomatic contralateral carotids) I showed that although the US plaque features were associated with symptom status, MRI-PH and a higher CAR score only one (Juxtaluminal Black Area - JBA) was predictive (OR =2.3 and 2.9 and 12.7, respectively). In the longitudinal study of 89 patients with a median of 905 days follow-up, the Greyscale Median (GSM) (log rank test: P=0.995) and JBA (log rank test: P=0.248) were not predictive of future cerebrovascular events. I found that the MRI-PH signal is generally stable over 24 months and MRI-PH negative showed in increase in hyperintense signal (P=0.020) with 7% becoming MRI-PH positive. In a cohort of 88 patients with > 50% carotid stenosis, there were 16 ipsilateral cerebrovascular events over a median follow-up of 1038 days. MRI-PH status predicted stroke (HR = 4.49 (95% CI 1.35-14.94, P=0.014). However the CAR score was not significantly predictive (HR = 1.272, 95% CI 0.410 – 3.951, P=0.677). Conclusions: This thesis shows that JBAs hold promise in being able to predict unstable plaques, but that in the small cohort study it was not predictive of cerebrovascular events. This is probably due to the small effect size and a low number of events recorded. Larger studies are required to properly test this finding. The MRI-PH is stable over a 2-year period and it may be associated with faster rates of stenosis progression. I found that MRI-PH is predictive of stroke in patients with >50% stenosis, but the CAR score is not.

Introduction: Medulloblastoma is an aggressive malignant neuro-ectodermal tumour of the cerebellum which accounts for 15-20% of childhood central nervous system tumours and frequently disseminates to the leptomeningeal spaces of the brain and spinal cord. Patients with disseminated disease respond poorly to post-surgical multimodal treatment which is thought to be explained by the intrinsic drug-resistant nature of these tumours. Here, we hypothesized that cells gain migratory and invasive capabilities by undergoing an Epithelial-Mesenchymal Transition (EMT)-like process whereby cells alter phenotypically and acquire stem cell-like properties during tumour dissemination. In this study, metastatic genes were identified and tested using 3D in vitro model systems which incorporated important components of the extracellular matrix. Using these markers, evidence for an EMT-like process and the role of the multi-drug transporter, ABCB1, was investigated in metastatic medulloblastomas. Small molecule inhibitors were also used to investigate whether metastatic processes could be targeted and drug resistance mechanisms could be circumvented in both in vitro and/or in vivo settings. Materials and Methods: Growth, morphology and biological processes (e.g. cell migration) were assessed in a panel of non-metastatic and metastatic medulloblastoma cell lines, as well as in non-tumourigenic neural stem cells cultured in a 3D basement membrane extract (BME) using the alamar blue assay (measure of metabolic activity) and time-lapse imaging. Putative metastatic markers were identified through literature review analysis of gene expression datasets or immunohistochemistry of tissue micro-arrays (TMA). These markers were then assessed in samples obtained from medulloblastoma cell lines cultured as 2D monolayers and grown in BME (for 3 and 6 days) by QRT-PCR analysis. Protein expression of selected markers were also assessed in mouse orthotopic xenograft samples by immunohistochemistry or in cell lines using immunofluorescence analysis. Finally, small molecule inhibitors (WIP1 and ABCB1 inhibitors) were used in 3D culture systems (3D spheroid and 3D BME assays) and in an orthotopic metastatic mouse model. Results: Medulloblastoma cell lines demonstrated different growth patterns in 3D. Metastatic cell lines formed metabolically active aggregates which sustained continual cell migration for at least 6 days; whilst non-metastatic and non-tumourigenic cells showed low metabolic activity and rapidly differentiated. Metastatic cell lines which were sustained longest in BME (D283 Med and MED1) demonstrated upregulation of the EMT transcription factor, TWIST1, along with several other EMT and TWIST1-related factors. Further analysis included overexpressing TWIST1 in a non-metastatic cell line (MED6 TWIST1) which induced a dispersed phenotype in 2D and cell aggregation in the 3D BME model which phenotypically resembled metastatic cell lines. TWIST1 and ABCB1 expression correlated with metastasis in patients and was upregulated in the invasive edge of primary tumours and in spinal metastases in an orthotopic metastatic mouse model. Small molecule inhibitors targeting WIP1 (a published metastatic marker) and ABCB1 inhibited cell migration of metastatic cell lines grown in 3D including the MED6 TWIST1 cell line. ABCB1 inhibition also increased sensitivity to etoposide treatment in 3D spheroid models and an orthotopic metastatic in vivo model. Conclusion: The 3D BME model utilised in this study can be used to distinguish metastatic capacity and transcriptional changes of medulloblastoma cell lines. Furthermore, data from this study supports a role for a TWIST1 driven EMT-like process in metastatic medulloblastoma and supports the use of ABCB1 inhibition to overcome chemoresistance.

Synaptic vesicles are the substrate of neurotransmission in most nerve terminals in the central nervous system. These small membrane spheres fuse with the synaptic membrane in an activity-dependent manner and release neurotransmitter into the synaptic cleft. Subsequently, vesicles are reclaimed through endocytosis prior to reuse. This recycling process is key to supporting ongoing signalling in the brain. While substantial effort has gone into defining basic characteristics of vesicle recycling, for example elucidating the timing of vesicle turnover, key questions remain unanswered. An important area with significant knowledge deficits relates to the relationship between vesicle function and ultrastructural organisation in the terminal. The aim of this thesis is to address this issue, exploiting new methodologies which provide novel insights into function-structure relationships of vesicle populations in acute brain slices. Specifically, this study considers organisational principles of three defined vesicle pools as well as examining the impact of an established plasticity protocol on pool properties. The first results chapter, Chapter 3, outlines and validates the novel protocol used for fluorescently labelling functional recycling vesicle populations in acute rat brain slices using the vesicle-labelling dye FM1-43 and new antibody based probes (syt1-Oyster, CypHer5E). Reporter-labelling and release properties are compared to similar approaches using cultured neurons. We conclude that this approach provides a more physiologically relevant method to study the functional properties of cells than used previously in cultured neurons. Chapter 4 outlines experiments utilising the capability of FM 1-43 to be photoconverted to an electron-dense form to allow a defined vesicle population, the readily releasable pool (RRP), to be characterised ultrastructurally. The RRP is arguably the most significant pool class, released first in response to an activity train. Functional assays and time-stamped electron microscopy are used to define basic properties of this pool, including its size, functional release kinetics, and temporal organisation. Specifically, the results demonstrate that retrieved vesicles are close to the active zone after stimulation, but mixed randomly in the terminal volume over 20 min. These findings address fundamental questions about vesicle reuse, the composition of future vesicle pools, and thus the mechanism of ongoing signalling in the brain. The same approach was used in Chapter 5 to examine the influence of Long Term Depression (LTD) on pool function and ultrastructure. LTD was induced in presynaptic terminals in CA1 via Schaffer collateral activation, and the following effects were observed: 1) a change in release kinetics; 2) a reduction in the total recycling pool size; and 3) no change in the composition of the docked pool. These findings demonstrate that there is a presynaptic component to LTD and that vesicle recruitment into the recycling pool appears to be an important possible substrate. However, the results suggest that such changes appear to be selective for specific pool subsets. Overall, work in this chapter offers new insights into fundamental principles supporting synaptic plasticity. Chapter 6 expands on previous studies which have demonstrated that recycling vesicles are constitutively shared between neighbours. This sharing of a ‘superpool' of vesicles has implications for the ability of synapses to adapt to changes in input weighting. In this chapter, the methods outlined above, as well as a new 3D EM technology, are used to define the size, positional organisation, and clustering properties of this pool in native hippocampal slice system. The findings in this chapter reveal that extrasynaptic vesicles appear to show a greater degree of motility than vesicles which remain in the intrasynaptic cluster, perhaps implying differential interactions with structural proteins in the synapse. Characterising the superpool is increasingly relevant, as it is now implicated in models of plasticity and disease. Taken together, these results show that the ultrastructural arrangement of recycling vesicles is highly activity-dependent, and that the cytoarchitecture plays a large role in determining the functionality of individual vesicles and synapses.

Primary Intracerebral haemorrhage is a severe form of stroke with poor prognosis attributed to haematoma characteristics. High blood pressure is present during the acute phase of intracerebral haemorrhage and associated with poor outcome in part through expansion of haematoma. Data from the ‘Efficacy of Nitric Oxide in Stroke trial’ (ENOS) was used to analyse the performance characteristics of qualitative and quantitative descriptors of intracerebral haematoma. The results showed that formal measurement of haemorrhage characteristics and visual estimates are reproducible. Intracerebral haemorrhage volumes measured using the modified ABC/2 formula were significantly lower compared to standard ABC/2 and computer assisted semi-automatic segmentation. In 629 patients with intracerebral haemorrhage presenting within 48 hours, the effect of blood pressure lowering with transdermal glyceryl trinitrate was assessed. Glyceryl trinitrate lowered blood pressure, was safe but did not improve functional outcome. In a small group of patients treated within 6 hours, glyceryl trinitrate improved functional outcome. Analysis of 246 patients with acute intracerebral haemorrhage from ENOS was undertaken to assess whether there were any differences in functional outcome among those who continued prior antihypertensive drugs during the immediate stroke period compared to those assigned to stop temporarily for 7 days. The results were neutral indicating that there was no benefit in those who continued treatment. Data of 1,011 patients with intracerebral haemorrhage in hyperacute trials from the VISTA collaboration showed differences in baseline characteristics and functional outcomes among patients from various ethnic backgrounds. A systematic review was updated to assess the effect of 26 randomised controlled trials that aimed to alter blood pressure within one week of acute stroke. The results showed that blood pressure reduction did not improve functional outcome irrespective of stroke type. When examined by time, treatment within 6 hours appeared to benefit but the number of patients were small and more studies are needed. The analysis also showed that continuing prestroke antihypertensive drugs in the immediate period after stroke did not benefit and might be harmful. In summary, this thesis provides new information on parameters used to estimate intracerebral haematoma, relationship between management of blood pressure and outcomes after haemorrhagic stroke. The work supports testing of whether very early blood pressure lowering after ictus is beneficial as is being undertaken in ongoing randomised controlled trials. Adjusting for ethnic differences may further identify patients in whom treatment may confer measurable advantage.

Sustained neural activity critically relies on the ongoing function of small central synapses. In particular, activity-driven fusion and recycling of neurotransmitter-filled vesicles at presynaptic terminals are key processes responsible for information transfer. Despite the fact that vesicle exocytosis and endocytosis are of great interest, the mechanisms of their regulation are still poorly understood. Moreover, hippocampal synapses exhibit high levels of variability in their structure and function, but the basis for this remains unclear. The aim of this work was to investigate these fundamental properties and establish key rules of regulation. Specifically, we wanted to test whether the timing of endocytosis of single synaptic vesicles was characteristic at individual boutons, and to investigate structural and molecular properties of synapses that underlie their particular behaviour. To explore this, we used a variety of optical imaging techniques in rat hippocampal neurons based on acutely applied probes such as FM1-43 dye, fluorescently tagged antibodies and genetically encoded reporters of presynaptic function, as well as ultrastructural readouts using electron microscopy. We found that although the timing of vesicle retrieval, measured with the optical reporter sypHy2x, was highly variable across the population of synapses, individual boutons showed signature endocytic kinetics. We also uncovered the properties of synapses that determine this behaviour, and demonstrated that these could be modulated, leading to predictable changes in the timing of recycling. These findings offer new insights into the rules that govern the function of presynaptic terminals. A second related objective examined was whether amyloid beta, the misfolding protein implicated in Alzheimer's disease, causes changes that are detrimental for efficient vesicle recycling. We showed that oligomeric amyloid beta 1-42 impaired endocytosis and disrupted other related presynaptic processes. We suggest that vesicle recycling mechanisms are important target substrates in Alzheimer's disease providing potential new avenues for development of therapeutic approaches.

Functional brain networks are interconnected brain regions that flexibly coordinate their activity to support cognitive demands (Fair et al., 2009). Functional brain connectivity describes a statistical dependency between the activities recorded at spatially distinct brain regions (Friston, 2009). Changes in the pattern of connections and level of activation in functional brain networks are thought to occur across development (Taylor, Donner, & Pang, 2012) but the nature of these changes and their relationship to cognitive development have yet to be delineated clearly. This thesis seeks to deepen our understanding of the development of functional brain connectivity across the age range 9-25 years. We used magnetoencephalography in conjunction with canonical correlation analysis to explore functional connectivity via amplitude-amplitude envelope correlations in 110 datasets (39 working memory, 33 relevance modulation (attention processing) and 38 resting state). At the core of this thesis, we have presented novel findings that show non-linear functional connectivity changes across development, with an increase from childhood (age 9-12) to late adolescence (age 17-20) followed by a reduction into young adulthood (age 21-25), resembling an inverted-U-shaped trajectory at least in the females included in this study. Whilst there are subtle yet statistically significant differences in how the functional connectivity profile from 1-100 Hz is modulated by different factors, the overall pattern of functional connectivity development appears to be remarkably consistent across cognitive demands and networks. Critically, this work is the first example of such findings and suggests that functional brain networks supporting higher order cognitive function are not alone in undergoing functional development; sensory networks that reach structural maturity early on in life also undergo functional development from age 9 to 25.

Stroke is a highly prevalent condition. It is a principal cause of adult disability and a leading cause of death in the United Kingdom (UK). The lifetime risk of stroke recurrence is also high, particularly for those with specific risk factors (e.g., hypertension). The long-term management of stroke is multi-faceted and complex. Stroke survivors are often responsible for self-managing their secondary preventive treatment (e.g., oral medication(s)) alongside rehabilitation, which is most beneficial in the first six-months. However, patients undergo a period of psychological adjustment after stroke. This is particularly evident in the early days where mood problems, such as depression, are highly common and affect how well people engage with, and adhere to, their rehabilitation and treatment. The present PhD research was interested in further understanding adjustment after stroke, and in particular, by utilising key evidence from the field of Health Psychology (more specifically, the Common Sense Model (CSM)), examining whether survivors’ perceptions (or beliefs) about their stroke are influential in determining how well people recover. The overall aims of this work were to: • Examine and synthesise the current evidence, through a systematic review and meta-analysis on the CSM in relation to physical illnesses to determine whether individual illness belief domains were important predictors of adherence to self-management in adults with acute or chronic conditions; • Elicit views regarding an assessment method (the Stroke Illness Perception Questionnaire-Revised (Stroke IPQ-R)) that would be suitable and acceptable to a stroke population for accurately measuring peoples’ beliefs in the acute phase (≤three-months post-stroke); • Apply the CSM (utilising the Stroke IPQ-R) to a population within the acute phase of stroke, to determine whether the model provides a useful framework for understanding survivors’ recovery after stroke (e.g., Health-Related Quality of Life (HRQL), disability, and mood). This research incorporated three standalone though inter-related studies, the first of which was a systematic review and meta-analysis. The key findings from this meta-analysis indicated that the majority of illness belief domains outlined by the CSM significantly predicted (albeit weakly) adherence to a range of self-management behaviours (e.g., attendance; medication adherence; diet; physical activity; and other disease-specific behaviours (e.g., blood glucose self-monitoring)). Pooled effect sizes ranged from 0.04 and 0.13. None of the relationships varied according to acute and chronic illnesses; the type of self-management behaviour; or the duration of follow-up. A qualitative study was subsequently undertaken. A variety of approaches were employed (including, expert consultation and Think-Aloud interviews) to develop a stroke-specific version of the IPQ-R. Interview findings showed very few problems with completion of the instrument. Where there were problems, these related to the wording of items, specifically abstract, complex and negative wording; the response format for the identity sub-scale; and questions on the timeline-cyclical and treatment control sub-scales. The revised scale was used in the third and final study. The final study examined the relationship of illness beliefs, assessed by the Stroke IPQ-R developed in study 2, and measures of post-stroke recovery, including HRQL, disability and mood. This was a longitudinal observational study involving 50 survivors (average age=66.9 years, standard deviation =14.5 years) within three-months of their stroke. The primary outcome for this study, HRQL, was measured using the EQ-5D-5L instrument. The secondary outcomes were: disability, which was assessed using the Barthel Index, Modified Rankin Scale, and Nottingham Extended Activities of Daily Living Scale; and mood, measured using the Patient Health Questionnaire-9. Various statistical approaches were employed in the analyses of these data, the results of which are as follows. Spearman’s correlations indicated that participants who perceived their stroke to have fluctuating effects and considerable distress at baseline ( < eight-weeks post-stroke) also reported worse mood one-month later. Multiple mediation analyses were conducted to examine whether baseline mood and coping (follow-up medication adherence, measured using the Medication Adherence Report Scale) mediated any of these relationships. The results indicated that baseline mood rather than coping was a significant mediator in the prediction of worse mood three-months post-stroke. Multiple linear regression analyses were carried out to determine whether baseline illness belief domains explained the additional variance in three-month HRQL, disability and mood, over and above that explained by socio-demographic (e.g., age, gender, and deprivation) and clinical variables (e.g., baseline co-morbidities, stroke severity, and indices of recovery). The findings indicated that baseline illness belief domains significantly added between 7.4 and 29.9% to the overall variance in models for the majority of the abovementioned outcomes. Last, the results from the study demonstrated that baseline illness belief domains were highly significantly inter-correlated at baseline, and with follow-up illness belief domains. In light of this, cluster analysis was carried out to explore whether stroke survivors in the acute phase could be grouped according to their illness belief schema. Three clusters were identified: ‘Low Adjusters;’ ‘Moderate Adjusters;’ and ‘High Adjusters.’ In contrast to ‘High Adjusters,’ Low Adjusters’ were participants who perceived their stroke to be chronic, with fluctuating effects, associated with a lot of symptoms, serious consequences and considerable distress. While baseline cluster membership was not significantly related to follow-up markers of post-stroke recovery, there were trends to suggest that ‘Low Adjusters’ had worse HRQL and mood, and greater disability one-month later compared to ‘High Adjusters.’ The present research had several important implications. The principal theoretical implication concerns treatment beliefs. The findings from all three studies highlighted that people’s beliefs about the effectiveness of their treatment in managing their condition are examined in the CSM in a limited way. There is currently a substantial focus on medication-taking behaviours despite treatments often being more complex (e.g., lifestyle behaviour or surgery). Therefore, the findings indicated that there is scope to further elaborate this domain, above and beyond what has already been undertaken in the Necessity and Concerns Framework. This examines the cost versus benefits decision-making that patients undergo when taking their medication(s) (Horne and Weinman 1999). In addition, suitable instruments need to be further adapted and validated to accurately measure these treatment beliefs. There were also several clinical implications of this work. First, it was emphasised that mood assessments should be carried out in patients immediately after stroke. It was shown in this research that even mild depressive symptomatology affected peoples’ beliefs about their stroke, and thus how well they psychologically adjust in the acute phase. Second, there is a potential to develop brief belief-based interventions for modifying maladaptive beliefs about stroke. For instance, addressing perceptions around the fluctuating effects of stroke and the considerable distress caused by stroke could form the basis for such interventions. In addition, interventions can be targeted to a particular type of stroke survivor (i.e., ‘Low Adjusters’), who are likely to be the people to benefit most from receiving them. However, these findings need to be borne out in a larger sample beforehand. The primary methodological consideration in relation to this work was the logistics of recruiting stroke survivors in the acute phase, which led to issues including: a small sample size; sample non-representativeness; and statistical constraints (e.g., factor analysis).