According to the European Brain Council, the annual total cost of brain disorders such as Huntington’s disease (HD) and Parkinson’s disease (PD) in Europe is approximately €386 billion. In order to develop therapies for neurodegenerative diseases, model systems that accurately reproduce the complex circuitry of the adult brain are needed. Neuron circuits developed in vitro could be used for studying pathogenesis of disorders and for high-throughput screening of potential therapies. In vitro models may offer the potential for highly reproducible and controllable cell circuitry, mimicking to some extent the complex neural connectivity required for function. Nano- and micro-scale substrates could be fabricated using techniques such as electrospinning, lithography and microfluidics to direct neurite orientation in order to build in vitro models that mimic in vivo circuitry. Poly-lactic acid (PLA) nano-fibres and polydimethylsiloxane (PDMS) micro-groove constructs were either pre-coated with poly-D-lysine (PDL) and laminin (LN) or pre-aligned astrocytes to study attachment and orientation of striatal neurites. Neurites were more responsive to substrates made up of combined topography and chemical cues; PDL and LN coated PDMS micro-grooves yielded the best neurite alignment. Excitability of striatal and cortical neurons was verified via an electrophysiology technique of patch clamp. PDMS microfluidic devices fabricated via lithographic techniques, were developed for co-culturing basal ganglia (BG) cells to model BG in vivo circuitry. Cell populations in the microfluidic device displayed electrical activity monitored using a calcium imaging technique. Connectivity was determined by eliminating activity of one cell population using tetrodotoxin (TTX) and studying response of remaining cell populations. Micro-contact printing was further explored as a technique for patterning BG cell circuitry instead of microfluidic devices. The microfluidic-based functional and complex model developed herein provides platform technology that can be useful for pharmaceutical and regenerative therapy and evaluation, therefore massively reducing costs currently associated with neurodegenerative diseases.

Neuronal cell loss and damage in the central nervous system are characteristics of debilitating brain related-degenerative disorders such as Parkinson’s disease. The last four decades of research have focused on the promise of cell replacement therapy to replace lost cells, repair the damage and provide functional recovery in affected neural circuits. A thorough understanding of the signals implicated in the development of neurons will greatly facilitate the use of cell replacement therapy. This project aimed to investigate the possibility of using nicotinamide, the amide form of vitamin B3, to promote the development of mature neuronal subtypes from mouse embryonic stem cells, and whether these could form a dopaminergic phenotype, to progress research in stem cell-derived therapies for Parkinson’s disease. Treatment of mouse embryonic stem cell monolayer cultures (46C Sox1GFP reporter cell line) with nicotinamide at the early onset of development not only increased the efficiency of neuronal generation but also enriched the ratio of purified neurons to non-neuronal cells. Nicotinamide acted at the initial stages of differentiation to promote accelerated neural lineage entry by embryonic stem cells in adherent monolayer cultures. The pluripotent stem cell and neural progenitor cell populations could be reduced by treating cells with nicotinamide, which also facilitated accelerated neuronal differentiation. Nicotinamide selectively enhanced the production of catecholaminergic, serotonergic and GABAergic neurons and, moreover, accelerated neuronal maturation. A reduction in the proportion of proliferating cells in nicotinamide-treated cultures was demonstrated– that is, nicotinamide enhanced cell-cycle exit, thereby promoting neuronal differentiation. The potential of nicotinamide was introduced to a novel, small-molecule-based strategy using pluripotent stem cell sources. Nicotinamide was shown to function synergistically with signalling molecules known to enhance a dopaminergic phenotype, to direct differentiating cells to adopt a dopaminergic cell fate. Thus, novel findings suggest that nicotinamide is a key signalling factor in brain development, and is required in a definable dosage range and times for the normal formation of dopamine neurons. This study supports previous evidence that vitamins and their metabolites play a fundamental role in neuronal development.

Background: Theories of motivational conflict are key in understanding alcohol misuse. Research suggests that approach and avoidance motivation are two distinct systems and that level of alcohol consumption depends on which system is most activated at one time. One factor thought to influence this balance is the role of implicit processes. This study aimed to investigate the effects of implicitly priming one motivational system (i.e. approach / avoidance) on the opposing system in regard to alcohol-related motivation in heavy drinkers. Methods: Heavy drinkers were recruited from a non-clinical community sample to complete a protocol of stimulus response compatibility and visual probe tasks designed to measure implicit motivation by recording reaction times to alcohol cues. Participants were assigned to one of three groups and attempts were made to manipulate implicit motivation using a masked priming paradigm. Measures of explicit attitudes towards alcohol were also administered. Results: No significant effects of priming were found. The overall sample showed attentional avoidance for alcohol cues presented at 50 ms duration but not at 500 ms. On the SRC task, participants were slower to avoid alcohol cues than neutral cues. Positive correlations were found between attentional bias for alcohol cues presented for 500 ms on the visual probe task and craving and consumption as measured by the Alcohol Approach Avoidance Questionnaire (AAAQ) and the Alcohol Use Disorders Identification Test (AUDIT) respectively. Significance: Implicit priming of alcohol-related motivational states had no influence on indices of alcohol approach and avoidance motivation or on attentional bias. As an overall sample, heavy drinkers showed automatic attentional avoidance of alcohol cues presented at short durations (50 ms). This is the first study to find avoidance of alcohol cues presented at this duration in heavy drinkers.

Background: There are multiple conflicting theories of depression and clients are frequently given contradictory explanations of their difficulties. Evidence that brings together biological, psychological and social factors of depression would be particularly useful in addressing this. The current study investigates the neural correlates of self-referential processing following psychological intervention for depression. This provides neurological evidence of how a central feature of psychological models may change following therapy. Methodology: Fourteen participants, who had received psychological intervention for depression, underwent functional magnetic resonance imaging scans whilst completing three types of cognitive task: a self-referential processing task, an other-referential processing task, and a graphical task. Participants’ neural activation during self-referential processing was compared to that of ten depressed participants and twelve control participants, which had been collected for a previous study. Results: When positive and negative self-referential processing were considered together, there was no normalisation of neural activation in the post-therapy group, despite normalisation on the BDI II. When positive and negative self-referential processing were considered separately there were fewer areas of significant neural activation during negative self-referential processing in the post-therapy group than in the depressed group. Indicating that neural activation in the post-therapy group normalised. In contrast, during positive self-referential processing, a lack of difference between the control group and the depressed group precluded the possibility of normalisation. Conclusions: The findings provide further support for the importance of the self in models of depression. In presenting neurological evidence in relation to psychological models and psychological therapy, they help bring together biological and psycho-social models of depression. It is possible that the ongoing patterns of atypical activation during self-referential processing represent a vulnerability to future episodes of depression. Possible explanations for the valence-specific findings are discussed and these are highlighted as interesting future research questions. Limitations of the research methodology are discussed and possible directions for future research are outlined.

Diffusion-Weighted Magnetic Resonance Imaging (DW-MRI) on the brain is a revolutionary method that provides in-vivo access to tissue macrostructure non-invasively (Basser et al., 1994). Recently, DW-MRI has been shown to have great potential in characterising brain microstructure, such as diameter and size distribution of neuronal fibres, features that were available so far only postmortem or through animal studies (Zhang et al., 2011). Using a process known as Tractography the existence of brain connections can be estimated using a set of DW images (Basser et al., 2000). The main aim of this thesis is to develop efficient methods for studying Tractography within a Bayesian framework. In order to characterise the white matter in the brain we focus on the widely used partial volume model (Behrens et al., 2003). We describe methods that are both time and computationally efficient for estimating the parameters of the partial volume model, before reparametrising the model, so that parameter estimation is viable in some special cases. The partial volume model allows for multiple fibre orientations so we develop methodology to choose between the number of white matter fibres in a voxel. We then take into account the uncertainty in the number of fibre orientations and provide a Fully Probabilistic Tractography method as an alternative to existing Tractography algorithms. Finally we look into the Global Tractography model (Jbabdi et al., 2007) and develop efficient methods for inferring connections between brain regions by investigating methods based on Thermodynamic Integration.

This thesis focuses on the care of people with dementia in long-term care settings. It considers both the experiences of staff working with people with dementia in such settings as well as the perceptions and satisfaction of family members of people with dementia who reside in long-term care. It is hoped that these papers will contribute to understanding how staff working with people with dementia in long-term care settings can be supported to improve the quality of care provision to this population. Chapter 1 is a systematic literature review investigating the staff factors which contribute to family satisfaction with ongoing care provision for their relatives with dementia who reside in long-term care. Empirical evidence from 14 articles was critically evaluated in order to identify relevant staff factors which contribute to family satisfaction. Relevant staff factors in three broad areas were found to contribute to family satisfaction with care provision for their relatives with dementia. Each area is explored and consideration is given to the implications for future research and clinical practice. Chapter 2 is an empirical study exploring the lived experience of healthcare assistants working with clients with dementia in residential care homes. Interpretative Phenomenological Analysis was used to analyse the data derived from semi-structured interviews with eight healthcare assistants. Three superordinate themes and nine subordinate themes emerged following the data analysis. Each theme is explored and consideration is given to the implications for future research and clinical practice. Chapter 3 is a reflective account of the researcher’s personal experiences of working with people with dementia. It explores the change in the researcher’s perceptions and attitude towards working with people with dementia as their knowledge and experience has increased over time and the impact that this has had on their outlook regarding working with people with dementia in the future.

Background Stroke is the foremost medical condition responsible for acquired disability and dependency. The initial psychological and physical deficits should arguably be identified early to allow interventions to be put in place if potential long-term sequelae are to be minimised. Physical impairments within stroke cohorts have been extensively researched and reported. We have access to numerous scales that describe general physical functioning during daily activities and its effects on independence and quality of life. Deficits in movement are easily identified and attributes of physical movements can be associated with such obvious measurement terms as strength, speed, co-ordination and task completion. As these attributes can be graded, it is simple to compare patients over time, within stroke cohorts and against the general population: controlled studies are straightforward, even if natural biological variation demands large samples. However, though physical impacts on a patient may be easily observed and measured, corresponding deficits in cognition and mood are less easily detected and quantified. Psychological problems are common within stroke populations, and exert both short and long term effects throughout all stages of rehabilitation. Despite our awareness of the potentially critical effects of psychological factors on patient outcome, there is a dearth of high quality research in this area. Although many cognitive and mood assessments are available, including some that were developed specifically for use in stroke, these are neither regularly administered nor have been convincingly shown to be accurate and reliable in identifying specific deficits. Thus, it is understandable that research available to us that describes prevalence and effects of cognitive and mood problems post-stroke is sparse in comparison to our knowledge of physical deficits. Methodology In order to begin to understand the effects that cognition and mood have on patient outcome post-stroke, a way of identifying prominent issues is required. Knowing that their effects can have both short and long term impact, reliable screening beginning early after stroke onset could offer opportunities to improve patient outcome through early implementation of interventions. Before an appropriate screening tool can be selected, evidence is required to support its feasibility and accuracy within acute stroke cohorts. In this thesis, I investigate cognitive and mood-screening assessments in stroke, across a series of linked projects. I carried out a review of the current research and I surveyed stroke units to identify which cognitive and mood assessments are commonly implemented. I collated and offer synthesis of the published data on accuracy of cognitive assessment instruments. I used these results to inform a diagnostic test accuracy study, examining selected measures that are commonly used in UK practice to screen for cognitive and mood problems. Based on these results, I designed (and was awarded grant funding for) a clinical study to assess test properties of cognitive and mood screening instruments in a rehabilitation setting and to describe potential obstacles affecting patient assessment. Findings There is heterogeneity in the choice of cognitive and mood tests employed across research and clinical practice. There was some overlap in assessment choice within these domains but no clear consensus on a preferred assessment tool. This is in part explained by the substantial number of tests available, it is telling that the most popular assessments accounted for only a fraction of the tool assessments employed. My literature based work also points to a relative lack of published science employing a cognitive or mood assessment tool. My review of diagnostic test accuracy found that properties of cognitive tools commonly used in practice and research (Folstein’s Mini Mental State Examination: MMSE, the Montreal Cognitive Assessment: MoCA, the Addenbrookes’ Cognitive Examination Revised: ACE-R and the Cambridge Cognitive Examination revised: R-CAMCOG) were susceptible to changing populations and purpose of assessment, with test properties differing when screening tools are used in acute and chronic stage of stroke. Depending on the cut-offs that are used to define “screen positive” cases, these tools would have varying ability to identify multi=domain cognitive impairment or dementia. Generally when applying standard (i.e. the traditional cut-off described for test use in an unselected population) cut-offs, sensitivity was good but specificity was low. Specificity could be improved when the cut-offs were altered while maintaining reasonable sensitivity and this suggests that screen positive thresholds may need to be altered to suit a stroke population The need for lowering our standard cut-offs suggests that there may be factors present in typical acute stroke patients which affect assessment accuracy compared to the populations and purpose for which these scales have been developed. Using the MOCA in the acute setting of my clinical study, confirmed that stroke cohorts require altered cut-offs to improve accuracy in cognitive impairment detection. A stroke cognitive assessment that can be derived from a standard neurological examination *the Cog4) has been described. Cross sectional comparison of MoCA and Cog 4 suggest that Cog4 has questionable validity and stroke specific cognitive measures are required since scores derived from other types of measures are not necessarily testing the most appropriate domains for stroke deficits. A lack of published data on cognitive and mood screening in the first days post-stroke suggested that describing the feasibility of assessing stroke patients in an acute setting would be a useful topic for research. My subsequent clinical study incorporated verbal and non-verbal assessments for mood and the MOCA. As well as usual test accuracy outcomes I considered feasibility issues such as proportion of patients suitable for initial approach, acceptance of assessment, prevalence of common stroke related impairments that mandate assistance or cause difficulty in completing assessments, or that preclude assessment altogether. A moderate proportion of patients who were approached declined to take part and several others required external assistance to complete the assessments. Shorter, less cognitively demanding assessments required less assistance and appeared to offer higher accuracy for predicting mood problems at follow-up. These results suggest that delaying cognitive and mood assessments until later during the post-stroke period may reduce the interference from acute stroke deficits. The final piece of work generated from my PhD studies, and that is ongoing, continues the theme of feasibility of cognitive and mood assessments. Cognitive and mood assessments are performed in stroke rehabilitation centres. The rehabilitation setting was chosen, as it will include varying patterns of physical and cognitive impairment. By comparing brief assessments and more lengthy measures of cognition, I hope to identify the most appropriate testing scheme that minimises patient burden. As part of this work I will describe the impact of stroke deficits on assessment and quantification of the patient’s psychological capabilities. Conclusions In conclusion, these studies have demonstrated a lack of guidance and of protocols for cognitive and mood assessment post-stroke. The evident heterogeneity in choices of assessment in research and usual practice indicated a need for evidence based accuracy studies. In conducting these I found that usual measures are susceptible to the population, timing, and cut-off used to define test positive cases, together indicating undesirable sources of variation. Transient stroke-related problems may lead to overestimation of persistent impairments. Although acute screening of cognition and mood would be possible, such screening may not be widely acceptable to patients and would require a high level of assistance from health professionals. Acute screening should only be performed if there are potential benefits that could impact on the patient from identification of cognitive or mood problems at this early stage. With the transient changes in cognition and mood that the majority of stroke survivors experience, screening is best left until later in the patient journey. However, there may still be potential feasibility issues of administration and assessment completion during later stages. Therefore, I suggest that studies that investigate what assessments are feasibly administered to stroke patients in later stages are required. This will inform future trial recruitment for complete data requirements as well as provide clearer picture of stroke survivors’ affected cognitive domains and or mood problems.

This thesis aimed to examine the roles of growth differentiation factor 5 (GDF5), a secreted member of the TGF-β superfamily of ligands with a well characterised role in limb morphogenesis, in the developing hippocampus and sympathetic nervous system. Previous studies have demonstrated that GDF5 promotes the growth and elaboration of dendrites from developing mouse hippocampal neurons in vitro and in vivo. As a first step to investigating whether GDF5 plays additional roles in the development of the mouse hippocampus, brains of P10 and adult Gdf5+/+, Gdf5+/bp and Gdf5bp/bp mice were analysed by anatomical MRI. The gross morphology and total volume of hippocampi were not significantly different between the three genotypes at either age, making it unlikely that GDF5 plays a significant role in modulating other aspects of hippocampal development in addition to promoting the growth and elaboration of dendrites. For this reason, no further time was spent on investigating whether GDF5 plays novel roles in regulating hippocampal development. Developing sympathetic neurons of the mouse superior cervical ganglion (SCG) require nerve growth factor (NGF) to promote their survival and target field innervation in vivo. Data in this thesis has revealed that GDF5 modulates NGF promoted survival and enhances NGF promoted process outgrowth in cultures of P0 SCG neurons. In addition, GDF5 promotes process outgrowth and branching from cultured perinatal SCG neurons in the absence of NGF. P10 Gdf5bp/bp mice, that lack functional GDF5 expression, display a marked deficit in sympathetic innervation of the iris, but not the submandibular gland, compared to P10 Gdf5+/+ mice. Whole-mount analysis of sympathetic innervation in P10 Gdf5bp/bp and Gdf5+/+ mice has revealed that GDF5 is required for correct innervation of the trachea and heart, but not the pineal gland. Further in vitro and in vivo investigations have suggested that the neurotrophic effects of GDF5 on developing SCG neurons are mediated by a receptor complex that includes the type 1 receptor, BMPR1A. These findings highlight a role for GDF5 in promoting the sympathetic innervation of selective target fields in vivo.

Background: Delusions are common in psychosis, defined as fixed, false beliefs. Some studies, however, have found that they may be less fixed than previously thought, possibly changing in response to talking about them. Relatives of people with psychosis or clinical staff often ask how to respond to them when they talk about their delusions, but no available advice appears to be evidence based. Aims: To review evidence on everyday communication about delusions and find out how people with delusions talk about them with others, taking three perspectives (patients, their nominated relatives and clinicians) and to construct a model for communication in relation to the delusion according to each party independently. Methods: 36 patients were engaged in semi-structured interviews about their mental state generally (Comprehensive Psychopathological Rating Scale) and their delusion (Maudsley Assessment of Delusions Schedule). Each patient was asked to nominate a relative and a professional to whom s/he spoke about the delusion. Relatives and staff were interviewed by different researchers. Results: Most patients reported speaking to others about their delusion and nominated an informant. Most felt emotionally disturbed by their delusions, but, against prediction, this did not affect nomination; nor did their delusion content. There was good agreement between the three parties on occurrence of such communication. Some patients had self-harmed; only some relatives or staff concurred with them on attributing this to the delusion. A testable hypothesis was generated that the intrusiveness of delusions resulted in personal change for the patient and sense of changed relationship and detachment for the others. Conclusions: No previous study has investigated communication about delusions between three parties. It was striking that so few relatives were engaged. If patients, their families and clinicians could improve mutual understanding of delusions, the safety of the patient and others as well as treatment might be improved.

Tumour necrosis factor (TNF) is an extensively well characterised proinflammatory cytokine. It is expressed as a type two membrane glycoprotein that is active both as a membrane-integrated ligand and as a soluble ligand following proteolytic release of the ectodomain from the cell membrane. TNF signals via two receptors, TNFR1 and TNFR2. In the immune system, it has been shown that these receptors can function as ligands for membrane-integrated TNF and initiate TNF reverse signalling. I was a member of a team that discovered, characterised and evaluated the physiological significance of TNF reverse signalling in the nervous system. We showed that TNFR1 is expressed in tissues innervated by sympathetic neurons and that this initiates TNF reverse signalling in postnatal sympathetic axons, which in turn enhances their growth and branching locally. Using a tissue whole mount method to visualize sympathetic fibres, I found that the innervation of multiple tissues that receive their innervation exclusively or predominantly from the paravertebral sympathetic chain is defective both in mice lacking TNF and mice lacking TNFR1. Sympathetic fibres reach these tissues in these mice but fail to grow and branch extensively in these tissues. In contrast, tissues that receive their sympathetic innervation predominantly from prevertebral ganglia are either unaffected, in mice lacking TNF and TNFR1, or hyperinnervated. Using live calcium imaging, pharmacological blockers of calcium channels and shRNA gene knockdown, I obtained evidence that T-type calcium channels are required for the effects of TNF reverse signalling on axon growth. I also showed that TNF reverse signalling enhances the growth of sensory axons, during an earlier stage in development than sympathetic neurons. This work establishes that TNF reverse signalling is widely involved in regulating axon growth in the developing peripheral nervous system.