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Timing behaviour plays an important role in the daily living of individuals from a great variety of species. For example, organisms must be able to discriminate between the durations of relevant events (temporal discrimination) and to regulate their own behaviour in time (temporal differentiation). The processes that allow animals to adjust their behaviour to the temporal regularities of the environment have been studied using different procedures which model the relationship between time and behaviour. A taxonomy of timing based on the subject’s location in time with respect to the signalled duration has been proposed. When an organism judges the duration of an elapsed interval the timing is retrospective (e.g. interval bisection); when it responds during an elapsing duration the timing is immediate (e.g. fixed-interval peak procedure); and finally when it chooses between future delayed outcomes the timing is prospective (e.g. inter-temporal choice schedules). It has been proposed that the cortico-striato-thalamo-cortical (CSTC) circuits play a special role in interval timing and inter-temporal choice behaviour. This thesis examined whether performance of timing tasks by rats induces neuronal activation within the prefrontal cortex and corpus striatum, as revealed by Fos expression, and explored a new approach to analyzing performance in an inter-temporal choice schedule. Chapter 1 describes the literature which forms the background of the project. It reviews interval timing and inter-temporal choice methodology and theory, the neurobiological substrates underlying both kinds of behaviour, and finally Fos expression, as a marker of neuronal activation. Chapters 2-4 present experiments that examined whether, in intact rats, performance of different interval timing tasks was associated with neuronal activation in the dorsal striatum and prefrontal cortex, as revealed by expression of the Fos protein, the product of the immediate-early gene c-fos (Experiments 1-3). Chapters 5-7 present experiments focused on some behavioural and neurobiological aspects of inter-temporal choice behaviour. One purpose of these experiments was to develop an abbreviated approach to estimate the rate of delay discounting (K) and reinforcer size sensitivity parameter (Q) based on the Multiplicative Hyperbolic Model of inter-temporal choice (MHM), using the adjusting-delay schedule. Additionally a novel way of quantifying transitional behaviour in the adjusting-delay schedule was presented based on analysis of the power spectrum of cyclical changes in the adjusting delay, dB (Experiment 4). This approach was used to analyze data obtained from rats performing on the adjusting-delay schedule under methodological manipulations (Experiment 5) and neurobiological interventions (Experiment 6). Experiment 1 (Chapter 2) investigated whether, in intact rats, performance on the discrete-trials temporal discrimination task was associated with neuronal activation in the prefrontal cortex and corpus striatum, as revealed by enhanced Fos expression in these areas. Performance on temporal and light-intensity discrimination tasks was well described by a two-parameter logistic equation. The rats trained under the timing task showed increased Fos expression in the orbital prefrontal cortex (OPFC) and the nucleus accumbens (Acb) compared to the rats trained under the light-intensity discrimination task, indicating a substantial activation of these areas during the timing task. However, there was no evidence for involvement of the dorsal striatum in the performance of this task. Experiment 2 (Chapter 3) examined whether performance on an interval-bisection task in the range of milliseconds showed increased Fos expression in the prefrontal cortex and corpus striatum compared to performance under a light-intensity bisection task. Performance on both bisection tasks conformed to the conventional logistic psychometric function. The rats trained under the timing task showed increased Fos expression in the OPFC, infralimbic and prelimbic cortex and Acb compared to the rats trained under the light-intensity bisection task. The results provided no evidence for an involvement of the dorsal striatum in the performance of this task. Experiment 3 (Chapter 4) investigated whether performance on the fixed-interval peak procedure (FIPP) was associated with increased neuronal activity in the prefrontal cortex and corpus striatum, as revealed by Fos expression. The results showed that response rate during peak trials was characterized by a ‘Gaussian plus ramp’ function, with maximal responding (peak rate) occurring around the time of the reinforcement in the FI trials (peak time). Consistent with the results of Experiments 1 and 2, the concentration of Fos-positive neurones in the OPFC was greater in rats exposed to FIPP than in rats exposed to a VI schedule. However, the results did not provide any evidence for a specific involvement of the dorsal or ventral striatum in FIPP performance. In Experiment 4 (Chapter 5), rats made repeated choices on an adjusting-delay schedule. Indifference delays, calculated from adjusting delays in the last 10 sessions, were shorter when the sizes of reinforcers were 14 and 25 µl of a 0.6 M sucrose solution than when they were 25 and 100 µl of the same solution. The ratio of the indifference delays (d50) was significantly smaller than that predicted on the basis of an assumed linear relation between reinforcer size and instantaneous reinforcer value. Estimates of K and Q fell within the values reported previously. Adjusting delays in successive blocks of trials were analysed using the Fourier transform. The power spectra obtained from individual rats had a dominant frequency that corresponded to a period of oscillation of the adjusting delay between 30 and 100 trial blocks. Power in the dominant frequency band declined with extended training. Experiment 5 (Chapter 6) examined the pattern of oscillation of dB in an adjusting-delay schedule using the power spectrum analysis. The step-size in which the delay to the larger reinforcer (dB) increased or decreased was tested across two conditions. In Condition 1, dB increased or decreased (according to the rats’ choice) by 20% from block n to block n+1. In Condition 2, the step size was 10%. The power spectrum analysis showed that the period of oscillation of the dominant frequency of the spectrum was significantly longer under Condition 2 than under Condition 1. There was a consistent trend for the power of oscillation to be higher in the initial segment of the experiment in both conditions. Experiment 6 (Chapter 7) examined the effect of excitotoxic lesion of the core of the nucleus accumbens (AcbC) on K and Q in an adjusting delay schedule using the same protocol as Experiment 4. The effect of the lesion on the power spectrum parameters was also examined. The AcbC-lesioned group showed significantly lower values of d50 than the sham-lesioned group. The ratio of the indifference delays seen in both groups was substantially less than the value predicted on the basis of an assumed linear relation between reinforcer size and instantaneous reinforcer value. K was higher in the lesioned group than in the sham-lesioned group; Q was not affected by the AcbC lesion. Neither the spectral power within the dominant frequency band nor the period corresponding to the dominant frequency differed significantly between groups. The final chapter (Chapter 8) summarizes the findings of the experiments, and discusses their implications for the putative role of the prefrontal cortex, and ventral and dorsal striatum in interval timing and inter-temporal choice, and for theoretical models of these behaviours. The role of the dorsal striatum is questioned, while a possible role of the Acb in temporal processing is proposed.

This thesis investigates the application of physiological measures made using magnetic resonance imaging (MRI) to study cerebral haemodynamics and the pharmacological modulation of brain activity. Blood Oxygen Level Dependent (BOLD) functional MRI (fMRI), together with Arterial Spin Labelling (ASL), were used to study of the effect of beta-blockers on the brain’s response to emotional visual stimuli at 3 T. The study aimed to test the James-Lange theory, which states that emotions result from the perception of bodily arousal. Autonomic nervous system responses to emotional stimuli can be predicted by the level of activity in the limbic system (including amygdala, brainstem and salience network). This thesis assesses the action of the peripherally acting beta-blocker, nadolol, on the fMRI response to neutral, pleasant and unpleasant visual stimuli, and during rest. 80 mg nadolol and placebo tablets were administered to twenty healthy human subjects in a double blinded, randomised, placebo controlled crossover design. Drug induced reduction in anterior insula response to emotional stimuli supports results from previously published studies, and the James-Lange theory. The preliminary finding of drug induced increase in amygdala response is contradictory. Territorial ASL (TASL) and multi-phase ASL were used in combination for advanced investigation of the vascular territories, and quantitative perfusion and blood transit times. These measures were used for the assessment of the location and haemodynamic properties of the cerebral watershed regions. Watershed region masks formed from TASL and multi-phase ASL data exhibited reduced perfusion and lengthened transit times compared to other cortical regions. The accuracy of watershed delineation was shown to be enhanced by the use of both techniques in combination. Furthermore, TASL is developed and implemented at 7 T. Alternative labelling schemes were compared and parameters optimised for control condition efficiency, and TASL assessment was performed in three healthy volunteers.

Introduction: Oxygen-glucose deprivation (OGD)±reperfusion and hyperglycaemia exacerbate the ischaemic cerebral injuries during or after a stroke. The key biochemical events associated with these pathologies include excessive cytoskeletal remodelling, modulation of tight junction proteins and the induction of oxidative stress. Recently, the overactivities of protein kinase C (PKC), RhoA/Rho-kinase, and pro-oxidant NADPH oxidase have been shown to account for the development of these events and the consequent disruption of human blood-brain barrier (BBB) integrity. Objectives: This thesis focused on the putative roles of RhoA/Rho-kinase signalling in OGD and OGD+reperfusion-evoked modulation of cytoskeletal remodelling, tight junction proteins and oxidative stress in human brain microvascular endothelial cells (HBMEC). The effects of hyperglycaemia-mediated PKC overactivities in modulating the RhoA/Rho-kinase pathway with reference to the aforementioned parameters i.e. cytoskeletal remodelling and tight junction protein expression and localisation have also been the focus of this thesis. Methods: For the OGD studies, the HBMEC were exposed to normoxia (controls), OGD (4, 20 hours) alone and followed by reperfusion (20 hours). The HBMEC-human astrocyte (HA) cocultures were established to mimic human BBB before exposing them to the experimental conditions. The integrity and function of HBMEC-HA cocultures were measured by transendothelial electrical resistance (TEER) and flux of permeability markers sodium fluorescein (NaF) and Evan’s blue-labelled albumin (EBA), respectively. For the hyperglycaemia studies, the HBMEC monolayers and the cocultures were exposed to normoglycaemia (5.5 mM D-glucose), hyperglycaemia (25 mM D-glucose), and hyperglycaemia with inhibitors of Rho-kinase, PKC, PKC-α, PKC-β, PKC-βII, PKC-δ; and the BBB integrity and function were measured by the TEER and flux studies, respectively. Fold differences in the protein expression or activity of RhoA, Rho-kinase-2, mono- and di-phosphorylated myosin light chain-2 (MLC2), total MLC2, gp91-phox (a pivotal NADPH oxidase subunit), catalase, occludin, claudin-5, zonula occludens-1 (ZO-1), β-catenin, and vinculin were either measured by in-cell or ordinary Western analyses. Results from the OGD studies: OGD compromised the barrier integrity as observed by decreases in TEER values and concomitant increases in flux of EBA and NaF across the cocultures. Transfection of HBMEC with constitutively active RhoA also decreased the TEER and increased the NaF paracellular permeability, whereas inactivation of RhoA by anti-RhoA-IgG electroporation exerted barrier protective effects. Moreover, OGD alone and after constitutively active RhoA transfection introduced stress fibres in HBMEC, which were abrogated by inactivation of RhoA and the specific inhibition of its main effector Rho-kinase by Y-27632. In addition, dramatic increases in the protein expressions of RhoA-GTP, Rho-kinase-2, gp91-phox, and antioxidant catalase were observed in HBMEC exposed to OGD+reperfusion conditions. These along with increases in the NADPH oxidase activity and total superoxide anion levels confirmed the oxidative stress in HBMEC under these experimental conditions. A marked rise in the protein expressions of claudin-5 and β-catenin observed after OGD (20 hours) alone and followed by reperfusion may represent the effects of oxidative stress on tight and adherens junction proteins stability, respectively. These results also concurred with marked decreases in TEER and concomitant increases in the flux of EBA across the in vitro models of human BBB exposed to OGD±reperfusion conditions when compared with the controls. Cotreatment with Y-27632 under OGD±reperfusion normalised the protein expressions of RhoA, Rho-kinase-2, gp91-phox, claudin-5, catalase; activities of RhoA and NADPH oxidase; and total superoxide anions levels, alongside improving the expression of occludin and the coculture integrity under the OGD±reperfusion conditions. Results from the hyperglycaemia studies: Hyperglycaemia also increased RhoA-GTP, Rho-kinase-2, mono- and di-phosphorylated MLC2 protein levels and total PKC activity. These changes were consistent with the actin stress fibre formations, ZO-1 and occludin redistribution from HBMEC periphery. Hyperglycaemia-mediated endothelial-barrier dysfunction was further characterised by reduction in TEER and elevation in flux of EBA. Glucose normalisation, RhoA neutralisation by anti-RhoA-IgG electroporation and Rho-kinase-2 inhibition by Y-27632 normalised all abovementioned protein expressions, restored actin and tight junction protein localisations and barrier integrity. Cotreatment of HBMEC with hyperglycaemia and a general PKC inhibitor namely, bisindolylmaleimide-I normalised the Rho-kinase-2, mono- and di-phosphorylated MLC2 levels. Moreover, specific inhibitors of PKC-α (Ro-32-0432), PKC-β (LY333531), PKC-βII (CGP53353) attenuated the PKC overactivity, normalised all protein expressions, restored actin localisation and improved barrier integrity. In addition, the PKC-α and PKC-β siRNA transfections mimicked the effects of the specific inhibitors and attenuated the hyperglycaemia-evoked RhoA-GTP, mono- and di-phosphorylated MLC2 protein levels and stress fibre formations. Conclusions: The RhoA/Rho-kinase overactivities compromise the endothelial-barrier integrity, in part, by modulating the cytoskeletal remodelling and inducing the NADPH oxidase-evoked oxidative stress under OGD±reperfusion pathology. Moreover, hyperglycaemia-mediated increases in PKC-α and PKC-β activities exacerbate the endothelial-barrier dysfunction by modulating RhoA/Rho-kinase signalling pathway. Summary: These findings support the hypothesis that OGD±reperfusion and hyperglycaemia perturb BBB integrity through regulation of RhoA/Rho-kinase activity and modulation of cytoskeletal reorganisation, oxidative stress and tight junction protein expressions or localisations.

Not only can stroke kill, but it can also disable and handicap the survivors. There is no medical treatment for stroke and not all stroke can be prevented. Rehabilitation, to promote recovery, or maintenance, to support those who do not recover, is required. Evidence about the efficacy of stroke rehabilitation is poor. There is little evidence to support many of the specific techniques used, but there is evidence to support the use of organised rehabilitation in hospitals. After leaving hospital there is some evidence that rehabilitation in out-patient departments and at home may be of further help. In this thesis, the results of a study undertaken to add to this slender body of knowledge by comparing domiciliary to hospital-based rehabilitation after hospital discharge are presented and discussed. Overall, no difference was found in terms of survival, institutionalisation, disability or perceived health between a domiciliary and a hospital-based rehabilitation service (day hospitals and out-patient departments). However, young stroke patients who had required considerable amounts of rehabilitation in a Stroke Unit, were best given further therapy at home rather than in out-patient departments, since it improved household and leisure abilities. This result is compatible with the only other controlled study of domiciliary stroke rehabilitation after hospital discharge. For frail elderly patients, the day hospital service may have had advantages over the domiciliary service because death and institutionalisation rates were lower. The latter finding may be spurious, due to allocation bias and small sample size. In view of the expense of day hospitals, more research is required to examine their efficacy. It is concluded that domiciliary rehabilitation is a small step forward for stroke rehabilitation and will benefit some disabled stroke survivors, and may be a more resource-efficient way of treating many others.

Background: High blood pressure is common in acute stroke and has been linked with poor outcome. Hence, outcome might be improved by lowering blood pressure. This thesis investigates the potential for glyceryl trinitrate, a nitric oxide donor, for lowering blood pressure in acute stroke. Methods: A systematic review was employed to clarify the relationship between outcome and BP in observational studies. Next two systematic reviews of animal studies using nitric oxide therapeutics in experimental stroke were performed to assess the effects on infarct volume and cerebral perfusion. Finally, two randomised controlled clinical trials of glyceryl trinitrate were performed in acute stroke patients to measure the systemic and cerebral haemodynamic effects. Results: In observational studies high blood pressure in acute stroke was associated with subsequent death, death or dependency, and death or deterioration. In experimental stroke nitric oxide sources and selective nitric oxide synthase inhibitors significantly reduced stroke volume. Glyceryl trinitrate lowered peripheral and central blood pressure and increased aortic compliance when given <48 hours from stroke. Glyceryl trinitrate did not alter quantitative measures of cerebral perfusion despite significantly lowering blood pressure <5 days from stroke. Conclusion: High blood pressure is a therapeutic target in acute stroke and animal data support the use of nitric oxide sources for lowering blood pressure. It is feasible to use glyceryl trinitrate for this purpose since it does not compromise cerebral perfusion. Trials now need to urgently assess the effect of lowering BP on outcome.

Part A: Memory rehabilitation is a promising approach to address memory difficulties although its effectiveness with neurologically impaired individuals is yet to be established (Chapter 1). This thesis was conducted within the context of the pilot and main phase of a randomised controlled trial (ReMind) evaluating the effectiveness of memory rehabilitation for people experiencing memory problems following traumatic brain injury (TBI), stroke and multiple sclerosis (MS). The trial compared the effects of restitutive and compensatory memory rehabilitation strategies with a self-help control intervention on memory functioning, mood, activities of daily living and mental adjustment. The quantitative data obtained in this trial did not provide strong evidence to support the effectiveness of the intervention. The use of inappropriate outcome measures may account for the contradictory or inconclusive findings of the ReMind and other memory rehabilitation studies. Chapter 2 provides a review of measures that were used in the ReMind trial and/or were commonly used to evaluate outcome in memory rehabilitation studies. A lack of measures that considers the aims of memory rehabilitation and the needs of neurologically impaired individuals was observed. The post-intervention interviews of participants (N=19) in the pilot phase of the ReMind were analysed thematically (Chapter 3). Participants reported benefits in areas that were not covered by existing quantitative outcome measures such as insight into the nature and severity of their memory problems, confidence in their ability to manage these difficulties and qualitative improvements in the use of memory aids. The aim of the following studies was to develop and evaluate a questionnaire responsive to the effects of memory rehabilitation following acquired brain injury. The process included two stages: Part B: Identification of the content of AMEDO questionnaire: At this stage, studies were conducted within the main phase of the ReMind trial. The content areas of the questionnaire were identified based on the input and feedback of participants in each of the three memory rehabilitation programmes (Restitution, Compensation and Self-help groups). A mixed methods design was followed and information was drawn from two sources: 1)Real time observations of 43 sessions were performed (Chapter 4). The study introduced a new recording strategy by using a time sampling method to qualitatively record the content of conversations. Group activity was also evaluated. Following a quantitative content analysis method, observations were grouped into categories and their frequency was assessed in order to systematically describe and compare the content of the three programmes. 2)Semi-structured post-intervention interviews were conducted with 20 participants to explore their experience in the groups (Chapter 5). Recurrent patterns of data were identified inductively following a thematic analysis approach. Interviews from each programme were analysed separately and the emerging themes were compared and contrasted to highlight similarities and differences between the programmes. The majority of participants perceived the main benefits of memory rehabilitation to be: a) responding to their need for information on the cognitive effects of brain injury, b) enhancing their sense of self-efficacy and control over their memory difficulties, c) motivating them to adopt a more proactive attitude towards the management of these problems. The advantages of the group based approach to rehabilitation were also highlighted by most respondents. Questionnaire items were generated to cover the key content areas that were identified in both studies: memory knowledge, awareness, emotional adjustment, active coping, control beliefs, attention, significant others (comprised Part A of the questionnaire), the use of external memory aids (comprised Part B1) and the use of internal memory aids (Part B2). Part C: Evaluation of the psychometric properties of Adaptation to Memory Difficulties Outcome questionnaire (AMEDO): The final study of this thesis (Chapter 6) evaluated the psychometric properties of the new questionnaire. The first version of AMEDO included 45 items rated on a 4-point Likert scale (strongly disagree-strongly agree). Face validity was assessed by researchers and clinicians experienced in the area of neurological rehabilitation. The psychometric properties of AMEDO were evaluated by posting the questionnaire to a sample of people with MS and TBI identified through hospital records. It was returned by 110 people with MS and 34 people with TBI and 87 of these participants returned the second questionnaire that was sent to assess stability. After applying the criteria of face validity, response distribution, and construct validity 15 items were retained in Part A of the questionnaire, and four items in Parts B1 and B2 respectively. The distribution of responses was acceptable for all items except for the ones assessing the use of external memory aids which displayed negative skew (ceiling effects). Principal component analysis indicated that the questionnaire captured most of the content areas it was designed to cover. Part B1 and B2 formed two distinct subscales assessing effectiveness in the use of external and internal memory aids respectively. Part A comprised three components: “Memory knowledge”, “Control” and “Emotional adjustment”. Evaluation of item-convergent validity confirmed the three factor solution. Internal consistency estimates for all the subscales were found to be satisfactory (α=.74-.84). Correlations between the subscales indicated that the represented constructs were related in meaningful ways. Test–retest reliability coefficients for the subscales ranged between r=.60 to r=.82. The biggest discrepancies were found in the “Memory knowledge” and external memory aids (EMA) subscales whereas the “Control” and “Emotional adjustment” showed acceptable levels of stability. Differences between test-retest scores in the former subscales dropped to satisfactory levels when analyses were repeated excluding respondents who completed the 2nd questionnaire more than a month after completing the 1st questionnaire. This finding indicated that the observed differences between scores at Time 1 and Time 2 may be reflective of actual changes taking place during that interval. Finally, it was shown that scores were not significantly affected by age, diagnosis and time since injury and, therefore, the questionnaire may be relevant to a wider neurological population. In conclusion, AMEDO is a brief and simple measure tailored to the characteristics and needs of neurologically impaired individuals. The questionnaire shows promise as an outcome measure specific to the effects of memory rehabilitation, to complement memory batteries and established generic measures. Further evaluation of the scale should confirm the stability of the questionnaire and assess its responsiveness to changes following memory rehabilitation.