The alpha-2 adrenergic receptor (α2-AR) subfamily includes three different subtypes, α2A-, α2B- and α2C-AR, all believed to exert their function through heterotrimeric Gi/o-proteins. The present study was undertaken in order to investigate subtype differences in terms of cellular response and to explore other potential signalling pathways of α2-ARs. Evidence is provided for a strong Gs-protein coupling capability of the α2B-AR, leading to stimulation of adenylyl cyclase (AC). The difference between the α2A- and α2B-AR subtypes, in this respect, was shown to be due to differences in the second intracellular loops of the receptor proteins. Substitution of the second loop in α2A-AR with the corresponding domain of α2B-AR enrolled the chimeric α2A/α2B receptor with functional α2B-AR properties. Dual Gi and Gs coupling can have different consequences for AC output. Using coexpression of receptors and G-proteins, it was shown that the ultimate cellular response of α2B-AR activation is largely dependent on the ratio of Gi- to Gs-protein amounts in the cell. Also Gi- and Go-proteins appear to have different regulatory influences on AC. Heterologous expression of AC2 together with Gi or Go and the α2A-AR resulted in receptor-mediated inhibition of protein kinase C-stimulated AC2 activity through Go, whereas activation of Gi potentiated the activity. α2-ARs mobilize Ca2+ in response to agonists in some cell types. This response was shown to depend on tonic purinergic receptor activity in transfected CHO cells. Elimination of the tonic receptor activity almost completely inhibited the Ca2+ response of α2-ARs. In conclusion, α2-ARs can couple to multiple G-proteins, including Gi, Go and Gs. The cellular response to α2-AR activation depends on which receptor subtype is expressed, which cellular signalling constituents are engaged (G-proteins and effectors), and the signalling status of the effectors (dormant or primed).

Amyloid beta (ABeta) and tau protein are both implicated in memory impairment in early Alzheimer’s disease, but whether and how they interact to cause synaptic dysfunction are unknown. Consequently, I firstly investigated whether tau protein is required for the robust phenomenon of ABeta-induced impairment of hippocampal long-term potentiation (LTP), a widely accepted cellular model of memory. I demonstrate that the absence of tau prevents the ABeta-induced impairment of LTP; moreover, a specific inhibitor of the tau kinase glycogen synthase kinase 3 blocks both an ABeta-induced increase in tau phosphorylation and the ABeta-induced LTP impairment. Thus, tau protein, likely in its phosphorylated form, is required for ABeta to impair LTP. Secondly, I investigated the underlying mechanisms for this ABeta-induced impairment and find that ABeta changes the balance between the two major types of glutamate receptors involved in plasticity processes, with a specific effect on GluN2B subunit-containing NMDA receptors. Since the distribution of these receptors is asymmetric between the left and right mouse hippocampus, I accessed these different types of synapses optogenetically and found that only the GluN2B-rich synapses receiving left CA3 input show ABeta-induced changes in the balance of glutamate receptors, suggesting an asymmetry in synaptic vulnerability to ABeta. Moreover, there was a left-right difference in tetanus-induced LTP and therefore, thirdly, I investigated whether mice have a hemispheric dissociation in memory processing using acute optogenetic silencing of left or right CA3 during hippocampus-dependent memory tasks. Unilateral silencing of either the left or the right CA3 caused a deficit in short-term memory, but only left CA3 silencing impaired performance on a spatial long-term memory task. Together, these results suggest that memory may be routed via distinct left-right pathways within the mouse hippocampus, and that neural pathways subserving distinct functions may also be differentially vulnerable to pathological changes at the synaptic level.

Fundamental to the complexity of the nervous system is the precise regulation in space and time of the production, maturation, and migration of neurons in the developing embryo. This is eloquently seen in the forming cranial sensory ganglia (CSG) of the peripheral nervous system. Placodes, which are transient pseudostratified neuroepithelia in the surface ectoderm of the embryo, are responsible for generating most of the neurons of the CSG. Placodal progenitors commit to the neuronal fate and delaminate from the epithelium as immature, multipolar neuroblasts. These neuroblasts reside in a staging area immediately outside the placode. Differentiation of the neuroblasts is intimately coupled to their adoption of a bipolar morphology and migration away from the staging area to the future site of the CSG. Thus the forming CSG is a highly tractable model to anatomically separate the three phases of a neuroblast’s lifetime: from neuroepithelial progenitor (in the placode), to immature neuroblast (in the staging area), to mature neuron (in the migratory stream). In this thesis, I used the forming CSG as a model to investigate the role of Regulator of G protein Signalling 3 (RGS3) in neuroblast commitment and differentiation. Promoters within introns of the RGS3 locus generate isoforms in which N-terminal sequences are sequentially truncated, but C-terminal sequences are preserved. Intriguingly, I found that expression of these isoforms in the forming CSG is temporally co-linear with their genomic orientation: longer isoforms are exclusively expressed in the progenitor placode; a medium isoform is expressed exclusively in the neuroblast staging area; and the shortest isoforms are expressed in the neuronal migratory stream. Furthermore, through loss- and gain-of-function experiments, I demonstrated that each of these isoforms plays a specific role in the differentiation state in which it is expressed: placode-expressed isoforms negatively regulate neurogenesis; the neuroblast-expressed isoform negatively regulates differentiation; and the neuron-expressed isoforms negatively regulate neuronal migration. The negative regulatory role which all isoforms play in different cell-biological contexts is intriguing in light of the fact that they all share a C-terminal RGS domain, which canonically negatively regulates G protein signalling. Through domain mutation and deletion, I showed that the RGS and N-terminal domains are important for the function of each isoform. Thus temporally co-linear expression within the RGS3 locus generates later-expressed isoforms which lack the regulatory N-terminal domains of the earlier-expressed isoforms, giving them new license to perform different biochemical functions. Lastly, I investigated the conservation and evolution of RGS3 and its isoforms. RGS3 was found to be present in all extant metazoans, and results from this thesis implicate it as the founding member of the R4 subfamily of RGS proteins. Furthermore, in the early vertebrate lineage, a critical domain was lost. This is intriguing in light of the fact that placodes in their stereotypic forms also emerged early in the vertebrate lineage. Ectopic overexpression of the full-length invertebrate RGS3 protein prevented pseudostratification of the vertebrate placode, suggesting that the domain loss in the early vertebrate lineage was important for the evolution of pseudostratified placodes and the expansion of the vertebrate nervous system. In summary, the work in this thesis has uncovered a previously unseen model of transcriptional regulation of a single locus: intragenic temporal co-linearity. Furthermore, the demonstrated functions of this regulation have profound implications on the generation and differentiation of vertebrate neurons, as well as the evolution of the vertebrate nervous system.

Diffusion-weighted magnetic resonance imaging can be used to estimate microstructural parameters of white matter in the brain. Two complementary techniques are investigated: the use of the temporal diffusion spectrum to explore small length scales and the STEAM technique to probe larger features. The diffusion spectrum has the potential to be more sensitive to small pores compared to conventional time-dependent diffusion. However, analytical expressions for the diffusion spectrum of particles only exist for simple geometries such as cylinders, which are often used as a model for intra-axonal diffusion. We propose a mathematical model for the extra-axonal space with parameters that are related to the microstructural properties of pore size, tortuosity, and surface-to-volume ratio. Measurements were made with an extra-axonal space phantom to validate the model. Fitted values for the phantom pore size match those from simulation. We extend the model to include the intra-axonal signal contribution. However, the parameters used to describe the intra- and extra-axonal spaces are related and it is important to remove redundant parameters to avoid overparameterization, which would make the model less robust. We propose analytical expressions to simplify the model. The model was then applied to measurements on fixed corpus callosum, which is a model system consisting of parallel axons. The estimated values of the axon volume fraction and mean and standard deviation of the axon radius distribution are comparable to those found in literature. Temporal diffusion spectra are useful for measuring the geometric properties of small spaces such as axon radii. However, longer diffusion times accessible using the STEAM sequence are necessary to probe structures with longer diffusion distances such as those parallel to the direction of axons. We used a model from the literature originally developed for use with animal magnetic resonance scanners and simplified it to quantify axial hindrance from data acquired on healthy volunteers in a clinical scanner. The interpretation of axial hindrance, which is a largely unexplored area of research, is discussed.

Turning while walking is a crucial component of locomotion; yet, little is known about how the biomechanics of turning gait differ from those of straight walking. Moreover, it is unclear how populations with restricted gait ability, such as children with cerebral palsy (CP) adapt to turning, compared to their typically developing (TD) peers. Thus, the aims of this thesis were to quantify the biomechanical differences between turning gait and straight walking in TD children and to explore if further, pathology specific, changes present during turning in children with CP. Biomechanical data, including three-dimensional body motion, ground reaction forces, and muscle activity from both groups were collected during straight walking and 90 degree turning gait using motion capture technology. Experimental data were used to compute joint kinematics (angles) and joint kinetics (moments and power) as well as more novel measures to quantify turning fluency and dynamic stability. These data were also used to derive walking simulations using a musculo-skeletal model of the human body in order to quantify muscle contributions to medio-lateral center of mass (COM) acceleration. The results show that both groups preferred to redirect their body during turning about the inside, rather than the outside, limb (with respect to the turn center). For TD children, substantial biomechanical adaptations occurred during turning, compared to straight walking. Furthermore, turning gait simulations reveal that proximal (hip abductors) and distal (ankle plantarflexors) leg muscles were mainly responsible for the redirection of the COM towards the new walking direction during turning. For children with CP, the results suggest that turning gait may be better able to reveal gait abnormalities than straight walking for a number of kinematic and kinetic gait variables. Potentially, analysis of turning gait could improve the identification and management of gait abnormalities in children with CP.

This thesis is concerned with the neurocircuitry that underpins the cardiovascular response to exercise, which has thus far remained incompletely understood. Small animal studies have provided clues, but with the advent of functional neurosurgery, it has now been made possible to translate these findings to humans. Chapter One reviews the background to the studies in this thesis. Our current understanding of the cardiovascular response to exercise is considered, followed by a discussion on the anatomy and function of various brain nuclei. In particular, the rationale for targeting the periaqueductal grey (PAG) and the subthalamic nucleus (STN) is reviewed. Chapter Two reviews the use of deep brain stimulation (DBS), in which deep brain stimulating electrodes are implanted into various brain nuclei in humans, in order to treat chronic pain and movement disorders. This technique not only permits direct electrical stimulation of the human brain, but also gives the opportunity to record the neural activity from different brain regions during a variety of cardiovascular experiments. This chapter also gives a detailed methodological description of the experimental techniques performed in the studies in this thesis. Chapter Three identifies the cardiovascular neurocircuitry involved in the exercise pressor reflex in humans using functional neurosurgery. It shows for the first time in humans that the exercise pressor reflex is associated with significantly increased neural activity in the dorsal PAG. The other sites investigated, which had previously been identified as cardiovascular active in both animals and humans, seem not to have a role in the integration of this reflex. Chapter Four investigates whether changes in exercise intensity affect the neurocircuitry involved in the exercise pressor reflex. It demonstrates that the neural activity in the PAG is graded to increases in exercise intensity and corresponding increases in arterial blood pressure. This chapter also provides evidence to suggest that neural activity in the STN corresponds to the cardiovascular changes evoked by the remote ischaemic preconditioning stimulus in humans. Chapter Five identifies the cardiovascular neurocircuitry involved during changes in central command during isometric exercise at constant muscle tension using muscle vibration. It shows that, in humans, central command is associated with significantly decreased neural activity in the STN. Furthermore, the STN is graded to the perception of the exercise task, i.e. the degree of central command. The other sites investigated appear not to have as significant a role in the integration of central command during the light exercise task that was undertaken. Chapter Six studies the changes in muscle sympathetic nerve activity (MSNA) during stimulation of various brain nuclei in humans. Regrettably, the results presented in this chapter are not convincing enough to support the hypothesis that stimulation of particular subcortical structures corresponds to changes in MSNA. However, the cardiovascular changes that were recorded during stimulation of the different subcortical structures are congruous with previous studies in both animals and humans. Chapter Seven presents a brief summary of the findings in this thesis.

Many phenomena in the BAEP are difficult to explain on the basis of the accepted hypothesis of its origin (after Jewett, 1970). The alternative mechanism of origin to which these phenomena point is summation of oscillations. Therefore, simulation of the BAEP by a mathematical model consisting of the addition of four sine waves was tested. The model did simulate a normal BAEP as well variations in the waveform produced by reversing click polarity. This simulation gives further clues to the origin of the BAEP. The four sine waves begin simultaneously; corresponding BAEP oscillations must, therefore, originate from a single structure. These oscillations begin in less than half a millisecond after the click. This suggests that the structure from which they arise is outside the brainstem. This alternative mechanism indicates that wave latencies do not reflect nervous conduction between discrete nuclei, and interpretation of BAEP abnormality need to be reconsidered. It also implies that mathematical frequency analysis is more appropriate, but this could be applied only when these methods have been perfected. Meanwhile, through visual analysis and recognition of oscillations, abnormality can be detected and described in terms that may have physiological significance.

Processing of visual stimuli in the vertebrate retina is complex and diverse. The retinal output to the higher centres of the nervous system, mediated by ganglion cells, consists of several different channels. Neurons in these channels can have very distinct response properties, which originate in different retinal pathways. In this work, the retinal origins and possible functional implications of the segregation of visual pathways will be investigated with a detailed, biologically realistic computational model of the retina. This investigation will focus on the two main retino-cortical pathways in the mammalian retina, the parvocellular and magnocellular systems, which are crucial for conscious visual perception. These pathways differ in two important aspects. The parvocellular system has a high spatial, but low temporal resolution. Conversely, the magnocellular system has a high temporal fidelity, spatial sampling however is less dense than for parvocellular cells. Additionally, the responses of magnocellular ganglion cells can show pronounced nonlinearities, while the parvocellular system is essentially linear. The origin of magnocellular nonlinearities is unknown and will be investigated in the first part of this work. As their main source, the results suggest specific properties of the photoreceptor response and a specialised amacrine cell circuit in the inner retina. The results further show that their effect combines in a multiplicative way. The model is then used to examine the influence of nonlinearities on the responses of ganglion cells in the presence of involuntary fixational eye movements. Two different stimulus conditions will be considered: visual hyperacuity and motion induced illusions. In both cases, it is possible to directly compare properties of the ganglion cell population response with psychophysical data, which allows for an analysis of the influence of different components of the retinal circuitry. The simulation results suggest an important role for nonlinearities in the magnocellular stream for visual perception in both cases. First, it will be shown how nonlinearities, triggered by fixational eye movements, can strongly enhance the spatial precision of magnocellular ganglion cells. As a result, their performance in a hyperacuity task can be equal to or even surpass that of the parvocellular system. Second, the simulations imply that the origin of some of the illusory percepts elicited by fixational eye movements could be traced back to the nonlinear properties of magnocellular ganglion cells. As these activity patterns strongly differ from those in the parvocellular system, it appears that the magnocellular system can strongly dominate visual perception in certain conditions. Taken together, the results of this theoretical study suggest that retinal nonlinearities may be important for and strongly influence visual perception. The model makes several experimentally verifiable predictions to further test and quantify these findings. Furthermore, models investigating higher visual processing stages may benefit from this work, which could provide the basis to produce realistic afferent input.

In type II diabetes, systemic metabolism is perturbed and on a cellular level the balance of fuel use is upset. More specifically, increased fatty acid use is seen alongside decreased glucose metabolism. This altered fuel use is mediated by changes in the activity and expression of multiple enzymes. One such enzyme within the glucose breakdown pathway is pyruvate dehydrogenase, whose activity is known to be reduced in the diabetic state. The field of real-time metabolic investigation has rapidly expanded over the past few years due to the invention of technology that has enabled the production of ¹³C labelled hyperpolarised compounds, which can generate high signal levels in magnetic resonance spectroscopy. This has provided the opportunity to measure real-time metabolism of injected hyperpolarised tracers both ex vivo and in vivo. This thesis aimed to develop the use of hyperpolarised compounds in vivo, to investigate the cardiac and hepatic metabolism of a diabetic rat model. We initially addressed the systemic nature of the disease by establishing a two-slice acquisition for obtaining cardiac and hepatic data during a single injection of hyperpolarised pyruvate. This was tested in the fed and fasted states before being used in the studies described in the subsequent chapters of this thesis. The value of hyperpolarised compounds in following metabolic modulation by drug treatment was explored in the next chapter. The effect on metabolism of two drugs targeted at pyruvate dehydrogenase, which differed in their isoform specificity, was investigated first in the perfused heart and subsequently in vivo, both in control and diabetic animals. Hyperpolarised magnetic resonance spectroscopy was combined with other established techniques to help both our understanding of the systemic changes that had occurred following treatment, and provide links between cardiac metabolism and function. The final chapter of this thesis explored the use of hyperpolarised ¹³C pyruvate to understand the effect of hypoxia on pyruvate dehydrogenase, firstly in healthy animals and subsequently in the diabetic, metabolically altered state. Understanding the combination of diabetes and hypoxia was interesting given the existence of several opposing metabolic effects seen in the two states. Overall this thesis has demonstrated developments in the use of hyperpolarised pyruvate that, when appropriately combined with other techniques, can yield valuable metabolic information, in terms of following disease progression, drug development, and understanding basic metabolism.

Neurogenesis continues postnatally in two major neural stem cell (NSC) niches: The subventricular zone (SVZ) and dentate gurus of the hippocampus. SVZ NSCs self-renew and produce transit amplifying progenitor cells that, in turn, divide and give rise to neuroblasts. These neuroblasts migrate to the olfactory bulbs, via the rostral migratory stream (RMS), where they terminally differentiate into mature neurons. The postnatal SVZ (pSVZ) is more gliogenic than its adult counterpart (aSVZ), contributing to robust postnatal astrocytogenesis and oligodendrogenesis in the surrounding brain parenchyma. Studies examining Galectin-3 (Gal-3) in the aSVZ showed it has functions in regulating neuroblast migration, microglial activation, oligodendrocytic differentiation, and angiogenesis. However, the role of Gal-3 in pSVZ lineage progression is unknown. This thesis aims to unravel the roles of Gal-3 in regulating pSVZ lineage progression, fate choices, and NSC activation. In doing so, the thesis tackles the molecular pathways possibly involved in mediating the effects of Gal-3. I found through co-immunoprecipitation that Gal-3 was bound to β-catenin and both proteins were co-expressed in the aSVZ. In addition, expression of Gal-3 and Wnt/β-catenin signalling were downregulated as SVZ cells progressed through the lineage and became migratory. I hypothesised that Gal-3 may regulate lineage progression through regulation of Wnt/β-catenin signalling. To explore this hypothesis, Gal-3 overexpression, knockdown or control plasmids were co-electroporated with a Wnt/β-catenin reporter into the SVZ of postnatal day two mice. I found lineage progression was not altered by Gal-3 overexpression. Surprisingly, contrary to evidence described in the cancer literature, Gal-3 overexpression reduced Wnt/β-catenin signalling. This was accompanied by an acute reduction in proliferation. Also, more cells expressed p27/Kip1 in the SVZ, and more cells migrated into the RMS, suggesting increased cell cycle exit. However, NSC proliferation and clonal neurosphere forming capacity were not altered by Gal-3 overexpression, indicating that NSC activation was not influenced by Gal-3. While olfactory neuronogenesis was not altered by Gal-3 overexpression, striatal astrocytogenesis was increased while oligodendrogenesis was dampened. Further experiments revealed phosphorylation of Smad proteins 1/5/8 was increased in vivo and in vitro after Gal-3 overexpression. These findings indicate that Gal-3 positively regulated BMP signalling in the SVZ, possibly contributing to Gal-3's pro-gliogenic effects. Taken together, this thesis supports a model whereby a subpopulation of Gal-3-responsive pSVZ cells reacted to Gal-3 overexpression by acutely exiting the cell cycle, and possibly through the same mechanisms, switched from oligodendrocytic to astrocytic fate. These cellular responses might have been brought about, at least partially, by acute suppression of Wnt/β-catenin and activation of BMP signalling. These novel findings emphasise the regulatory actions of Gal-3 on pSVZ lineage progression through Wnt/β- catenin and BMP signalling.