This thesis comprises of three studies investigating the repair of brachial plexus avulsion injury from three different perspectives. The first aim of this thesis was to investigate the long-term effects of reimplantation surgery in patients with complete brachial plexus avulsion injury using standardized assessments. Patients are assessed with clinical examination, neurophysiological studies and patient-reported outcome questionnaires. It is shown that patients who have undergone brachial plexus re-implantation surgery demonstrate small, yet significant, improved function in motor and sensory recovery compared with patients who have not had this surgical intervention. These results are encouraging, but functional improvements are limited. One strategy aimed at further improving the effects of re-implantationis the transplantation of OECs during the surgical repair. The second study presented in this thesis comprises of a prospective observational study of human biopsies of nasal mucosa by endonasal dissection of the mucosa of the nasal septum during the approach for routine sinus surgery. Samples are cultured in the laboratory, and the yield of olfactory ensheathing cells is compared as to the location, size, and weight of the biopsies and patient characteristics including age, smoking, nasal disease severity. OEC yield is associated with mucosal disease and patients age. The third aim of this thesis is to develop novel MRI techniques that can be used in human trials of cell mediated repair of the brachial plexus to assess patients’ spinal cord regeneration after OEC transplantation and provide a more robust outcome measure for comparing different strategies of brachial plexus repair. We focus on magnetic resonance spectroscopy and demonstrate that this technique is sensitive to pathological changes that occur in the spinal cord above the injury. Myo-Inositol to creatine ratio is correlated with disability and is negatively correlated to time from injury. The implications of the above findings are discussed.

The molecular and genetic mechanisms involved in the pathogenesis of primary dystonia were investigated. This was carried out with two core studies. The pathogenesis of DYT1 dystonia was examined by the creation of a cell model. A single amino acid deletion in the DYT1 gene, which codes for a protein called torsinA, is responsible for most cases of primary, early-onset, generalized torsion dystonia. The gene in its wildtype and mutant forms were transfected into HEK 293 cells and the proteins locations and associations were studied. Wildtype torsinA was found to reside in the endoplasmic reticulum (ER). Mutant torsinA, however, was found in the nuclear envelope (NE) and in perinuclear inclusions that were not associated with the endoplasmic reticulum. In SH-SY5Y cells these inclusions were found to be associated with vesicular monoamine transporter type 2 (VMAT2), a transmembrane protein responsible for packaging catecholamine into vesicles. This association was studied in greater detail by immunoprecipitation and the functional implications were investigated by dopamine release experiments. The ultrastructure of the inclusions was also investigated by electron microscopy. Cervical dystonia and blepharospasm are the most common forms of focal primary dystonia. Most cases are apparently sporadic, although familial cases have been described. An allelic association study of genes involved in dopamine neurotransmission was carried out in order to determine whether genetic factors might contribute to an individual’s susceptibility to developing dystonia. An allele near the dopamine receptor 5 (DRD5) gene was found to be associated with cervical dystonia and blepharospasm. The presence of a causative mutation in the DRD5 gene was investigated by restriction fragment length polymorphism (RFLP) analysis and by sequencing.

The importance of sex differences in the brain and behaviour is indisputable. It forms the basis for differences in risk across a range of neurological and psychiatric disorders, as well as gender roles within society. The classical approach to investigating sex differences primarily involves comparisons between males and females. While informative for characterizing the wide array of sexually dimorphic traits, straight comparisons are insufficient to elucidate specific molecular contributions due to the multiplicity of confounding factors. Discrete genetic polymorphisms can be used to investigate variance in these traits due to sex-related molecular factors independent of confounds of sex. This thesis applies candidate genetics to understand the specific contributions of molecular components of the sex hormone pathways to sexual dimorphism in brain structure, personality and cognition. A cohort of 384 individuals were recruited to undergo MRI brain scans, cognitive and personality testing. They also provided blood samples for candidate genotyping in polymorphisms in genes for the androgen receptor, oestrogen receptors, progesterone receptor and aromatase enzyme that converts testosterone to oestrogen. Voxel-based morphometry was used to characterise regional differences in brain volume accounted for by these polymorphisms and the relationship to sex differences in brain volume. Diffusion tensor imaging was then used to determine variation in white matter integrity and structural connectivity due to these polymorphisms. Sex differences in personality and cognition are further investigated in terms of correlations with the polymorphisms and brain structure. Finally an endophenotype approach was used to investigate differential risk for conditions such as Alzheimer’s disease and depression between sexes through related brain and personality-based traits. The neural and molecular genetic mechanisms underlying this risk are inferred from correlations with brain-based measures and genotype. The strengths and weaknesses of this approach and the scientific implications of this work to gender research are discussed.

This thesis investigates a systematic approach to the use of MRI-guided and MRI-verified deep brain stimulation (DBS) in clinical practice. The concept of individual targeting of visualised brain structures without microelectrode recording (MER) was examined with respect to accuracy, safety and efficacy. Accurate MRI localisation of the pedunculopontine nucleus, an investigational new DBS target for parkinsonian gait disorders, is described and proof-of-principle confirmed in a cadaver study using MR-microscopy and histological examination. The impact of surgical trajectory on stereotactic accuracy in routine clinical practice was examined at two centres using MER: trajectories involving the ventricle suffered from significantly greater targeting errors compared to those that did not (p<0.001) and multiple brain-passes were more likely to be required to reach the intended target (p<0.01). Subcortical brain shift between pre and postoperative stereotactic images was minimal after MRI-verified procedures without MER (136 procedures); the observed shift did not adversely affect targeting accuracy or clinical outcome. A simple calibration process improved mean targeting errors by 0.6 mm (p<0.001) to 0.9 ± 0.5 mm from the intended target point. A large patient series was compared to a systematic literature review to determine factors associated with haemorrhage; an image-guided and image-verified approach carried a significantly lower risk of haemorrhage and associated permanent deficit than other surgical methodologies (p=0.001). Another study confirmed that, when observing certain precautions, cranial MR images could be obtained with an extremely low risk in patients with implanted DBS hardware. Outcome data from patients undergoing MRI-guided and MRI-verified surgery for Parkinson’s disease and primary generalised dystonia compared favourably to reports from the literature. Mode of anaesthesia did not impact on surgical outcome. In conclusion, this thesis demonstrates that a meticulous approach to MRI-guided and MRIverified DBS is safe and accurate, with clinical outcomes comparable to other techniques.

Risk is a highly salient psychological decision variable, and sensitivity to risk is an evolutionarily ancient attribute. In this thesis I address the neurobiological foundation of risk assessment, and show that behaviour is driven by an underlying distributed neural representation of different elements of risk in the brain. In particular, I show using fMRI (in Chapter 4) and MEG (in Chapter 8) that variance (dispersion) and skewness (asymmetry) of gambles evokes anatomically separable neural responses in a parietal, prefrontal and insula cortical network. I discuss possible theoretical neurobiological mechanisms by which preferences could be imbued to choice, and show that subjective tastes for risk, in terms of behavioural sensitivity to each of these risk dimensions, influences the encoding of risk and subsequent anticipatory responses. In Chapter 5, I show that a representation of prospective outcomes several trials into the future is supported by a dissociated encoding of the statistical information of future states in medial prefrontal cortex; furthermore that this encoding is contingent upon overarching goals or constraints. In Chapter 6, I demonstrate that economic choice is highly susceptible to exogenous biological influences, namely the effect of metabolic state, whilst in Chapter 7 I provide evidence that the encoding of risk is not affected by dopaminergic disruption, suggesting that dopamine might mediate effects on risk-taking via its role in reward feedback representation. In summary, the studies in this thesis elaborate the neural mechanisms underlying how humans make both single-shot and sequential decisions under risk, central elements in decision-making scenarios ranging from foraging to financial investment. This demonstrates that phylogenetically ancient circuitry subserving valuation and reward decompose choice into their salient statistical features, enabling the sophisticated representation of the future and its alternatives.

Although the tau gene (MAPT) is not mutated in the majority of tauopathies, there are pathological disturbances in tau-isoform homeostasis. Investigation into MAPT linkage disequilibrium and haplotype structure, and the common variation of MAPT associated with increased risk of progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) identified a possible basis for the disturbances in tau-isoform homeostasis. Of multiple haplotypes in the H1 clade, the H1c sub-haplotype drives the association with PSP and CBD, suggesting that it carries the pathogenic variation leading to increased risk of these largely sporadic disorders. The aim of this project was to investigate the regulation of the transcription of MAPT by first identifying transcription factors (TFs) and TF complexes that bind to the MAPT core promoter and its conserved regulatory domains. The specific aim was to determine if differential binding of TFs and TF complexes to the allelic variants of the SNPs in the MAPT promoter region forms the basis of the allele-specific differences in MAPT transcription and splicing that we have observed in vitro and in vivo. Results from electrophoretic mobility shift assays, pull-down experiments and mass spectrometry had shown potentially novel proteins binding to the disease-associated SNP under investigation. Bioinformatic analyses helped to stratify these proteins according to possible relevance. While immunoblotting alongside co-transfection of siRNA and luciferase promoter construct experiments had shown initial evidence of a complicated relationship between cis and trans factors within the tau promoter region. These findings were novel and they provide new insight into the regulation of one of the most important genes in neurodegeneration. The identification of MAPT transcriptional machinery would provide further insight into the role of MAPT in neurodegeneration and the basis for therapeutic intervention.

The primary aim of the research I have undertaken is to better understand the influence of dopamine on behavior and to build on knowledge of the various roles of dopamine in the healthy brain but also to improve understanding of the deficits affecting patients with Parkinson’s disease (PD), the hallmark of which is dopamine depletion. By testing PD patients on cognitive and motor tasks, we are able to probe the effects of dopamine depletion in humans. Testing PD patients in different medication states also provides a method with which to attempt to tease apart the various roles of dopamine from each other. My first two experiments use the PD model to this end whereas the third experiment utilises a pharmacological manipulation in healthy individuals. The aim of my first experiment was to tease apart the relative contribution of dopamine to learning from its influence on action performance, and by doing this to better understand the deficits which have been observed in PD patients in reinforcement learning tasks. The second experiment focuses on the motor deficits observed in PD. The aim of this study was to test whether these motor deficits can at least in part explained by the deficits in reward sensitivity. The third and final experiment in this thesis uses a pharmacological manipulation in healthy individuals to isolate the role of dopamine in set shifting in the context of a response to cues with negative hedonic valence, with the hope of better understanding the neurobiology underlying pathological behaviours associated with the hyperdopaminergic state.

The experiments presented in this thesis were designed to elucidate the distributed neuronal networks that support speech perception. In contrast to much of the published literature on this topic, I have chosen to do this by building and testing biologically informed models of how neuronal populations might interact to process speech stimuli. I took this approach because I believe that to gain a deeper understanding of brain function it is important to move from a topographic to a mechanistic level of description; in other words to move from asking, “Where in the brain are stimuli processed?” and move towards asking, “How does the brain process stimuli?”. The technique I have used to do this is called Dynamic Causal Modelling (DCM). This technique involves building models of how networks of neuronal populations might interact with each other to generate signals that are observable via neuroimaging methods, and to test these models against real data recorded from subjects performing tasks in the scanner. The first experiment applies dynamic causal modelling to magnetoencephalography (MEG) data to investigate the processing of meaningful speech sounds (phonemes). The second experiment applies this technique to functional magnetic resonance imaging (fMRI) data to investigate processing of the auditory, phonemic and lexicosemantic information present in speech. The results of both experiments are used to specify a novel, hierarchically organised network model of how the brain perceives and understands the sounds and meaning of speech. The final two experiments examine the functional consequences (at the behavioural and neuronal levels) of structural damage to the brain. This is done using dynamic causal modelling of fMRI data recorded from aphasic patients while they listen to speech. The results of these experiments show that structural damage to the auditory processing network identified in normal subjects results in widespread dysfunction throughout the entire speech processing network, and a significant decrease in the ability to understand the meaning of speech.

Mutations in the shaker potassium channel (KV1.1) gene KCNA1 are associated with episodic ataxia type 1 (EA1) while mutations in the CACNA1A gene which encodes the CaV2.1 calcium channel underlie episodic ataxia type 2 (EA2). Both disorders exhibit dominant inheritance and are characterised clinically by paroxysmal attacks of cerebellar dysfunction. This thesis explores the relationship between six new mutations in KCNA1, EA1 phenotype and functional consequences of expressed mutations. Expression of all six mutations demonstrated a loss of function. There was an imperfect correlation between phenotypic severity of EA1 and in vitro electrophysiology with respect to mutations. Supporting this indirect relationship, this thesis reports that non-genetic factors influence the phenotypic outcome of EA1 in monozygotic twins. In recognition of the importance of studying mutations in physiologically relevant conditions, this thesis describes the use of a lentivirus as a tool for studying the effects of KCNA1 mutations in neurons. A number of individuals with clinical EA2 do not harbour point mutations in CACNA1A. A screen for rearrangements in eighteen such individuals identified deletions of at least one exon of CACNA1A in three patients with EA2 suggesting that such large deletions are an important cause of EA2. This thesis tested the hypothesis that genetic variation in CACNA1A conferring a loss of CaV2.1 function is associated with the syndrome of episodic ataxia with epilepsy. CACNA1A was sequenced in 17 individuals with a clinical phenotype of EA accompanied by epilepsy and/or epileptiform EEGs. The functional impact of newly identified missense mutations and all non-synonymous coding variants (nCVs) in the 17 individuals was determined using voltage clamp experiments. Twelve of the 17 patients had genetic variants which reduced CaV2.1 function. These findings suggest that variations in CACNA1A which result in varying degrees of loss of channel function are likely to predispose individuals to episodic ataxia with epilepsy.

Transplantation of olfactory ensheathing cells (OEC) is one of the most promising current approaches to repair spinal cord injury. The encouraging results from transplantation of OECs in animal models have led to several clinical applications of these cells in spinal cord injury. The first controlled clinical trial was carried out by Mackay-Sim, Féron and colleagues (Mackay-Sim et al., 2008). A number of neurosurgical teams have also implanted foetal OECs (Huang et al., 2003) or minced whole mucosal tissue (Lima et al., 2006) into spinal injuries. So far the reported functional benefits are only moderate. The Mackay-Sim team reported no improvements while others reported minor improvements (including an ongoing trial by Pawel Tabakow’s team in Poland; personal communication). The basic conclusion is that OEC transplantation is feasible and safe. However, in the studies where suspensions of OECs were used there were not enough cells to fill the lesion, and no materials were used to bridge the gaps. In order to progress to more effective transplants the two areas addressed in this thesis will be important – what is the best source of adequate numbers of cells, and what biomaterials can be used to bridge the gaps. In addressing the twin necessities of (a) identifying the tissue source needed to provide sufficient cells for transplantation and (b) the problem of bridging the large gaps present in spinal cord injuries, the results of this study were directed towards two issues. (a) The questions of cell source and proliferation were addressed by establishing the quantitative baseline for the yield of primary cultures from the olfactory bulb, and the whole and split olfactory mucosa and characterising the heterogeneity of these cultures in search for any difference between bulbar and mucosal OECs. The study of flow cytometric simultaneous antigenic bivariate cell cycle of purified OECs and ONFs from these four sources revealed the evolution of population heterogeneity and its strikingly differences between these four sources of primary tissue with additional populations that were not previously described. An unexpected highly proliferative p75+ population in the stripped mucosal epithelium was also characterised. Correlation study of the cell proliferation and population evolution revealed cell autonomous among the difference sources. (b) The feasibility of a synthesis biomaterial for the deployment of OECs and olfactory nerve fibroblasts (ONFs) as a transplant was addressed by designing and developing an electrospun PLGA nanocomposite nanofibre construct with a myriad of microfabrication techniques, focusing on how OECs and ONFs can be deployed during tissue culture and transplantation. The techniques included nanocomposite electrospinning, replica moulding from photolithographed silicon mould, design of tissue-culture membrane insert, and laser ablation. The biocompatibility study showed that when grown on a fibre mesh structured at the nano-scale, OECs responded by adopting the elongated form comparable to that which occurs when the convey regenerating fibres cross small lesions in in vivo transplants. Preliminary functional studies of using the nanocomposite nanofibers as a neural scaffold in the organotypic entorhino-hippocampus slice co-culture data provide an indication that the nanofibres are compatible with tissue and allow migration of astrocytes and growth of nerve fibres. These observations will be important in future attempts to derive larger cell populations for transplantation. The anticipated use of the OEC nanofibre prosthesis would be in the application of autologous human OECs for bridging the gap in spinal cord lesions.