Neurodegenerative diseases are characterized by progressive degeneration of selective neurones in the nervous system and the formation of protein inclusions in surviving neurones. The mechanisms underlying neurodegeneration and neuroprotection in the nervous system remain elusive. Ubiquitin is one of the hallmarks of neuropathological inclusions in the majority of neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Therefore, dysfunction of the ubiquitin proteasome system has been implicated in disease cause and/or progression. This thesis investigates a unique conditional genetic mouse model of neurodegeneration caused by conditional genetic 26S proteasomal depletion in mouse forebrain neurones. We have identified potential proteins targeted for ubiquitination in brain using bio-affinity chromatography of zinc finger protein ZNF216 coupled with mass spectrometry. This lead to the identification of several potential ubiquitinated proteins involved in gene expression and regulation. We have also investigated the global brain proteome in response to 26S proteasomal depletion in neurones using two-dimensional fluorescence difference in-gel electrophoresis coupled to mass spectrometry for protein identification. Several differentially expressed proteins were identified in the 26S proteasome-depleted cortex. Astrocytic intermediate filament proteins glial acid fibrillary protein and vimentin, as well as the antioxidant peroxiredxoin-6, were upregulated. Mitochondrial fumarate hydratase and stathmin-1, involved in the tricarboxylic acid cycle and cytoskeletal microtubule dynamics respectively, were downregulated. These proteins have been validated by biochemical and immunohistochemical approaches. Further analysis of oxidative stress revealed increased lipid and protein oxidation that may be involved in the neurodegeneration associated with 26S proteasomal depletion. However, we also show increased phospholipase A2 activity associated with peroxiredoxin-6 expression that may have additional roles in neurodegenerative and/or neuroprotective functions. Interestingly, the levels of reactive oxygen species were inversely correlated with the upregulation of peroxiredoxin-6. We suggest that peroxiredoxin-6 may play an important role in the brain in the protection against oxidative stress and our studies may improve our physiological and pathological understanding of neurodegenerative disease.

The work presented in this thesis is intended to contribute towards the understanding of the cerebral vascular behaviour in response to changes in neuronal activation. The blood oxygenation-level dependent (BOLD) functional magnetic resonance imaging (fMRI) signal provides an indirect measure of neuronal activation, arising from a combination of metabolic and vascular (blood flow and blood volume) changes local to the activation. Therefore the BOLD signal is dependent on local vascular properties as well as on the neuronal activation, leading to a variability of the BOLD signal, based on the underlying vascular structure. It has become an important goal to improve understanding of the mechanisms underlying the BOLD signal in order to separate out this vascular variability from the underlying correspondence with the neuronal activation. The effect of field strength on the temporal characteristics of the BOLD haemodynamic response function is investigated. An earlier BOLD response onset has been measured with increasing static magnetic field strength, consistent with an earlier microvascular (compared with macrovascular) signal response. This result can be used to improve haemodynamic models of the BOLD signal. Hypercapnia, a vasodilator, has been used both to assess the relationship between transverse relaxation and blood oxygenation at 3 and 7 Tesla and to identify vascular heterogeneity between two equivalent brain regions. A tight, linear relationship was found between the level of hypercapnia and transverse relaxation at both 3 and 7 Tesla, whilst the change in transverse relaxation due to hypercapnia increased 2.1 ± 0.5 fold from 3 to 7 Tesla, indicating an approximately linear relationship across field strength. In a separate experiment, a vascular asymmetry was found between the left and right precentral gyri using hypercapnia. This result highlights the need to account for the vascular contribution to the BOLD signal before using this BOLD signal to make comparisons of neuronal activity across brain regions. Finally, an improved model for calibrated BOLD has been proposed and implemented, which requires fewer assumptions than existing approaches. This uses the BOLD response to some task, repeated both at normoxia and hyperoxia. To assess the validity of this model, the effects of paramagnetic oxygen molecules are considered, both dissolved in blood plasma and in airspaces adjacent to the brain. These effects were found to be small, except for in the frontal cortex.

The standard memory consolidation model assumes that new memories are temporarily stored in the hippocampus and later transferred to the neocortex, during deep sleep, for long-term storage, signifying the importance of studying functional and structural cortico-hippocampal interactions. Our work offers a thorough analysis on such interactions between neocortex and hippocampus, along with a detailed study of their intrinsic dynamics, from two complementary perspectives: statistical data analysis and computational modelling. The first part of this study reviews mathematical tools for assessing directional interactions in multivariate time series. We focus on the notion of Granger Causality and the related measure of generalised Partial Directed Coherence (gPDC) which we then apply, through a custom built numerical package, to electrophysiological data from the medial prefrontal cortex (mPFC) and hippocampus of anaesthetized rats. Our gPDC analysis reveals a clear lateral-to-medial hippocampus connectivity and suggests a reciprocal information flow between mPFC and hippocampus, altered during cortical activity. The second part deals with modelling sleep-related intrinsic rhythmic dynamics of the two areas, and examining their coupling. We first reproduce a computational model of the cortical slow oscillation, a periodic alteration between activated (UP) states and neuronal silence. We then develop a new spiking network model of hippocampal areas CA3 and CA1, reproducing many of their intrinsic dynamics and exhibiting sharp wave-ripple complexes, suggesting a novel mechanism for their generation based on CA1 interneuronal activity and recurrent inhibition. We finally couple the two models to study interactions between the slow oscillation and hippocampal activity. Our simulations propose a dependence of the correlation between UP states and hippocampal spiking on the excitation-to-inhibition ratio induced by the mossy fibre input to CA3 and by a combination of the Schaffer collateral and temporoammonic input to CA1. These inputs are shown to affect reported correlations between UP states and ripples.

The G1uN3 subunits of the NMDA receptor are thought to reduce the Ca 2+ permeability and Mg2+ sensitivity of NMDA receptors. cRNA for rat NMDA receptor subunits were injected into Xenopus oocytes and responses were recorded using two electrode voltage clamp at -100, -75 and -50 mV. G1uN1-1a/2A, GluN1-1a/2A/3A and G1uN1-1a/2A/3B containing receptors were characterised using Mgz+, memantine, philanthotoxin-343, methoctramine and MK-801. IC50 values were calculated and generally showed significant increases between those containing G1uN1-1a/2A/3 subunits and G1uN1-1a/2A, while those with G1uN3B were found to be significantly higher than G1uN3A. Activity was also typically shown to be partially restored with mutations at the N and N+1 site asparagines of G1uN3A. As the ICS0 was only partially restored the changes cannot be attributed to the loss of the N-site alone. Further differences may be due to a constricted threonine ring within the M3 vestibule region, or due to continued reduced flux through the channel. Another possibility is that to restore block it may require both the double N and N+1 mutation at the N-site. Multi-target-directed ligands combine two pharmacophores to produce drugs which retain the properties of the constituents. Memantine has been approved for use in Alzheimer's disease and there is a search for drugs that have similar actions. A range of multi-target compounds were tested to determine if NMDA receptor blockade activity was obtained. The pharmacophores explored were tacrine, donepezil, lipoic acid carvedilol and dimebon. The most promising compounds were carbacrine(3) (tacrine and carvedilol) and lipocrine (lipoic acid and tacrine), and it was found that the former was equipotent and the latter more potent than memantine. Potency was likely due to the tacrine moiety. These compounds should be further categorised to determine if they retain the kinetics that gives memantine its favourable side effect profile.

Single doses of phenobarbitone decreased the turnover rate of dopamine (DA) and noradrenaline (NA) and increased the whole brain levels of 5- hydroxytryptamine (5-RT) and gamma-aminobutyric acid (GABA) . Habituation to phenobarbitone increased the levels of DA in striata and midbrain and decreased that of cerebral hemispheres, leaving the total amount unchanged. Habituation resisted the depletion otherwise caused by alpha-methyl-p-tyrosine (alpha-m.p.t.) in the striata for DA and in the cerebral hemispheres for both DA and NA. Withdrawal of phenobarbitone decreased the levels of DA in the striata and both catecholamines in the cerebral hemispheres. Withdrawal increased the depletion of DA in the striata and cerebral hemispheres and that of NA in the cerebral hemispheres and midbrain caused by alpha-m.p.t. Withdrawal convulsions increased the levels of DA in the striata and decreased it in the cerebral hemispheres, leaving the total amount unchanged. NA was less in this group than it was in controls. Alpha-m.p.t. protected animals from convulsions. This group showed less DA in the striata and in the cerebral hemispheres and less NA in midbrain. Habituation to phenobarbitone increased the levels of 5-HT and 5-HIAA. Withdraiwal returned both levels to control values. While withdrawal convulsions decreased the levels of 5-HT, 5-HIAA levels were increased. Single doses of phenytoin increased the levels of DA in striata and NA in midbrain. It also increased the l levels of 5-HIAA in whole brain and decreased the depletion of 5-HIAA caused by p-chlorophenylalanine (p-c.p.a.) or pargyline. Long term administration of phenytoin increa sed the levels of dopamine in the striata and the midbrain and decreased that of the cerebral hemispheres. It also produced an increase in the level s of NA in the cerebral hemispheres. Similar effects were observed after alpha-m.p.t. Whole brain levels of 5-HT and 5- HIAA were increased after long term treatment with phentoin. Single doses of carbamazepine increased the levels of NA in the midbrain and decreased the depletion of 5-HIAA after p-c.p.a. and pargyline. The long term treatment with carbamazepine increased the total brain levels of 5- HT and 5-HIAA and those of DA in the striata and cerebral hemispheres and for NA in the cerebral hemispheres and midbrain. The same effect was seen after alpha-m.p.t. While NA and GABA levels were decreased in the primary focal area oneweek after cobalt implantation, 5-HIAA levels were increased. The same effect was seen for NA and 5-HIAA levels two weeks after cobalt implantation.

Odour Binding Proteins (OBPs) are found in the olfactory system of a range of species. Whilst invertebrate OBP function is well understood, the exact function of these proteins in the vertebrate nasal mucus is not fully understood. Multiple subtypes of rat OBPs have been identified and found to share less than 30% sequence identity. Studies have suggested each rat OBP binds to particular sets of odours, which may afford them a particularly important role within the olfactory system, pre-sorting odours. This study focuses on OBP3, closely examining the binding interaction of this protein with a range of odours. This has been done using Isothermal Titration Calorimetry which revealed that the binding of the highest affinity ligands, the heterocyclic compounds, is enthalpically driven. A defined odour series, the gamma-lactones showed that despite increasing ligand size and hydrophobicity, the free energy of binding of these ligands is maintained. Interactions with both 2-isobutylthiazole and the gamma-lactones were examinedusing NMR spectroscopy, which required the NMR assignment of OBP3 to be determined. In addition a homology model of OBP3 was created in order to structurally map the per-residue changes of OBP3 upon binding. It has been found that OBP3 is able to subtly adjust in order to accommodate each of these ligands. Protein engineering of the OBP3 binding pocket has been used to highlight the importance of its size and hydrophobicity. The importance of a tyrosine residue that appears to cover the opening to the binding pocket and is conserved across both the aBPs and the lipocalins family they are part of, has been demonstrated. Mutagenesis has also revealed the importance of a number of key residues for the binding of 2-isobutylthiazole. The ability to rationally improve the affinity of OBP3 for a particular odour has also been demonstrated.

Electrical stimulation of the heel or toes evokes short latency polysynaptic reflexes in muscles of the ankle extensor medial gastrocnemius (MG), the ankle flexor tibialis anterior (TA) and the knee flexor biceps femoris (BF), the co-ordinated actions of which form an organized protective withdrawal response. Previous studies in the rabbit have shown that such reflexes are enhanced (sensitized) or inhibited by application of the chemogenic agent mustard oil (MO) to various areas of the body surface in a manner that reinforces the protective function of these responses. The organization of these ‘sensitization fields’ was strictly controlled by supraspinal pathways from the brain. The aim of the present experiments was therefore to extend these studies of the spatial organization of sensitization of withdrawal reflexes into the rat, the species most commonly used in pain research. Patterns of facilitation and inhibition of spinal reflexes were obtained and compared in decerebrate spinalized, decerebrate non-spinal, and Alfaxan- anaesthetized rats by applying mustard oil to sixteen different body locations including sites on the ipsilateral and contralateral hindlimbs as well as other off limb areas such as the snout and tail. It was found that in decerebrate spinalized animals, MO application to ipsilateral hindlimb sites enhanced but never inhibited reflex responses in the limb, whilst MO treatment to off limb sites was without effect. In contrast in anaesthetized animals the prevalent effect of MO was inhibition from treatment sites distributed across the entire animal. Reflexes in animals with an intact spinal cord (decerebrate or anaesthetized) were facilitated or inhibited by MO application to ipsilateral hindlimb sites in a way that resembled the modular organization of reflexes per se and previous sensitization studies in the rabbit. However clear differences were also observed in the effects of MO between the two species, including modulation of the heel-MG extensor response in spinalized animals, which in rabbit was inhibited by MO application to the ipsilateral toes whereas in the rat no inhibition by MO was found in spinalized animals. Sensitization of hindlimb reflexes by MO in the rat therefore seems to be influenced by descending inhibitory and facilitatory pathways. These influences were further investigated in subsequent studies. Whilst the predominant effect of spinalization was a loss of inhibition and an expansion of sensitization fields, in the toes-evoked TA reflex the reverse was noted with regard to MO treatment of distal ipsilateral sites. In this case, facilitation found in non-spinal animals did not occur in the equivalent spinalized cohort, and thereby implies that a descending facilitatory pathway is also implicated in the control of spinal reflex excitability in this model. In decerebrate rats, the noradrenergic α2-adrenoceptor antagonist RX 821002 or the serotonergic 5-HT3 receptor antagonist ondansetron were administered directly to the spinal cord (intrathecally, i.t.) either alone (dose-response studies) or as a single dose between two successive MO applications to one of three ipsilateral skin sites on the hindlimb (heel, metatarsophalangeal joints or flexion of the ankle). Cumulative i.t. doses of RX 821002 revealed the presence of tonic descending inhibition of all reflex responses as well as preventing MO-evoked inhibition (and possibly facilitation) of reflex responses suggesting the involvement of the α2-adrenoceptor subtype in mediating these effects in this model. On the other hand, cumulative i.t. ondansetron administration resulted in a decrease in the magnitude of reflex responses, thus indicating that 5-HT3 receptors are indeed implicated in tonic descending facilitation of spinal reflexes. In addition i.t. ondansetron revealed that potentiation (and possibly inhibition) of reflexes following an acute chemogenic insult appears to involve the actions of serotonin at 5-HT3 receptors in the spinal cord. These studies therefore show that the organization of sensitization of hindlimb reflexes in the rat are modulated by supraspinal influences that exist as a balance of descending facilitatory and inhibitory pathways, mediated at least in part by serotonergic 5-HT3 receptors and noradrenergic α2-adrenoceptors.

Perceptual learning is an important mechanism in the human visual system, and can lead to long-lasting improvements across a broad range of perceptual tasks. In this study we demonstrated how perceptual learning can be applied to improve word recognition in the peripheral visual field of a sample of older individuals. We have shown that improvements in thresholds can be equalised across age, simply by increasing the number of training sessions available to older observers. Based on this initial finding we further sought to establish a protocol to induce improvements in reading ability for a sample of individuals with age-related macular disease (AMD). As a prelude to this work, we investigated the effects of crowding and fixation instability on similar tasks. Having suffered damage to their central vision, our target population (individuals with AMD) must use peripheral vision for daily viewing tasks. Peripheral vision is known to be highly susceptible to crowding, the influence of which has previously been shown to strengthen with age. We investigated the relationship between age and crowding on a letter recognition task, and found that (for this task) crowding was age in-variant, implying that this key inhibitor to peripheral visual perception should not have an inordinate influence on learning in our AMD sample. Our work on fixation stability also led to promising results. We demonstrated that our proxy for fixation instability (a dynamic target or dynamic fixation point) did not adversely affect letter recognition thresholds. Fixation instability is a common issue in AMD, but our data suggests that this may not adversely affect learning on our word recognition task. The final part of this work has been the implementation of a small study in which we trained a sample of individuals with AMD on our word recognition task. Significant improvements in thresholds were observed, though these did not quite reach the level of an age-matched normally sighted sample. Nonetheless, the trajectory of the learning curve suggests that further improvements would be possible with extended training sessions. Crucially, we also observed significant transfer of learning – from the trained word recognition task to an untrained sentence reading task (the MNRead Acuity chart). This is a key aspect of the study, as we are primarily interested in developing training protocols that lead to real-world improvements in visual ability. Improvements on MNRead scores are promising, and suggest that our approach may prove to be a useful starting point in the development of a robust therapeutic protocol.

Understanding the modulation of contextual fear learning and memory by the neurotransmitter dopamine is important as it could lead to a greater understanding of the mechanisms underlying anxiety disorders. The effect of D1 receptor antagonism during the contextual fear learning and memory stages was investigated. In the first set of experiments the D1 receptor antagonist SCH 23390 (0.1mg/kg; i.p.) was administered systemically before or immediately after contextual fear conditioning to determine whether D1 receptors are involved in the acquisition and/or consolidation stages. This experiment was followed up by investigating the effects of SCH 23390 infusion into the dorsal hippocampus (5μg per side) or amygdala (2.5μg per side) on contextual fear acquisition. The second set of experiments investigated the involvement of systemic SCH 23390 in the reconsolidation, retrieval, destabilization and extinction of contextual fear. SCH 23390 was administered before or immediately after either a short reactivation or longer extinction session. In the destabilization experiment SCH 23390 was administered prior to reactivation and the NMDA receptor antagonist MK-801 (0.1mg/kg; i.p) immediately after to determine if SCH 23390 could rescue the amnesic effects of NMDA receptor antagonism. It was found that systemic and intra-hippocampal but not intra-amygdala SCH 23390 reduced freezing during memory retention testing, twenty four hours and seven days after conditioning. There was no effect of SCH 23390 when immediately given after conditioning. There was also no effect of SCH 23390 when given either before or after reactivation or extinction sessions. The destabilization experiment was inconclusive as MK-801 was not found to impair memory when administered after reactivation. In conclusion, D1 receptors were found to be involved in the acquisition of contextual fear, and this modulation was found to occur in the dorsal hippocampus but not the amygdala. D1 type receptors were not found to be involved in the consolidation, retrieval, reconsolidation or extinction of contextual fear.

Background: Osteoarthritis (OA) is the most common musculoskeletal disease worldwide and a major cause of chronic pain. Treatment of OA pain is still suboptimal due to limited efficacy and considerable side effects of available analgesics. Pain in OA has a significant central component. Cyclooxygenases (COXs) and glial cells in the spinal cord and the periaqueductal gray (PAG) play a significant role in central sensitisation and pain modulation. Vascular endothelial growth factor-A (VEGF-A) is a key molecule in normal and pathological angiogenesis. Serine arginine protein kinase 1 (SRPK1), which phosphorylates serine arginine splice factor 1 (SRSP1), controls VEGF gene alternative splicing. This results in two splice variants; VEGF-A165a, which is pro-angiogenic and pro-nociceptive, and anti-angiogenic VEGF-A165b, which showed anti-nociceptive effects in models of neuropathic and inflammatory pain. Objective: This thesis investigated changes in COX expression and glial activation in the spinal cord and the PAG in the monosodium iodoacetate (MIA) model of OA. It also addressed the effects of VEGF-A165b and SRPK1 inhibitor SPHINX-31 on pain behaviour and joint pathology, as well as COX expression and glial activation in the spinal cord and the PAG in the same model. Hypothesis: VEGF-A165b and SPHINX-31 can prevent and/or reverse enhanced pain behaviour in the MIA model of OA, through involvement of spinal glial cells and COXs in the PAG and spinal cord. Vascular-astrocyte association in the PAG is enhanced in the MIA model of OA, and this effect is reversed by administration of SPHINX-31. Methods: Rats received an intra-articular injection of MIA (1 mg) in the knee. In one study, animals were treated with VEGF-A165b (i.p. 20 ng/g body weight twice weekly) on days 0-13 (VEGF(d0-13) group) or days 14-28 (VEGF(d14-28) group) after MIA injection; in another study, rats received SPHINX-31 (i.p. 0.8 µg/g body weight twice weekly; (MIA/SPHINX group)) for 19 days after induction of the model. Pain behaviour was monitored throughout the studies, at the end of which (day 28) tissues were collected for the assessment of joint histopathology and the evaluation of spinal COX-2 mRNA expression by PCR. In addition, immunofluorescence (IF) was used to assess COX-2 expression and glial activation in the spinal cord, as well as astrocyte activation and vascular-astrocyte association in the PAG. Results: VEGF-A165b significantly attenuated weight bearing asymmetry (%) in MIA rats on day 28 (29.58 ± 1.803 in MIA/VEGF(d0-13) group vs. 22.95 ± 2.088 in MIA/PBS group, p < 0.01; 29.23 ± 1.49 in VEGF(d14-28) vs. 22.95 ± 2.088 in MIA/PBS, p < 0.05). VEGF-A165b reversed mechanical withdrawal thresholds to the naïve level, but without reaching statistical significance. No significant changes in knee joint pathology were observed in VEGF-A165b treated MIA rats compared to the MIA/PBS counterparts. In the MIA/VEGF(d0-13) group, contralateral deep laminae of the dorsal horn had a higher percentage (%) of non-neuronal cells expressing COX-2 than the corresponding superficial laminae (3.26 ±1.16 vs 1.12 ± 0.43, p < 0.05), while no difference was observed in the MIA/PBS group. Administration of VEGF-A165b did not significantly affect spinal microglia and astrocyte activation, nor COX-2 expression in the PAG. SPHINX-31 had no significant effects on pain behaviour, joint pathology or spinal COX-2 expression in the MIA model of OA. On the other hand, MIA/SPHINX group exhibited a higher activation of spinal microglia than MIA controls (% of CD11b +ve cells in MIA/SPHINX-31 vs MIA/vehicle groups: 6.36 ± 0.89 vs 1.72 ± 0.38, p < 0.01). In addition, SPHINX-31 significantly increased astrocyte activation in the ipsilateral dorsolateral (DL) PAG relative to corresponding ventrolateral (VL) PAG (GFAP IF intensity: 12.89 ± 1.52 vs 8.46 ± 0.84, p < 0.05), and it increased vascular-astrocyte association (%) in the contralateral DL PAG relative to corresponding VL PAG (70.35 ± 7.68 vs 38.92 ± 8.19, p < 0.05). Interestingly, naïve rats had a significantly higher astrocyte activation and vascular-astrocyte association in the VL PAG than in DL PAG. Conclusions: VEGF-A165b exerted a significant antinociceptive effect in the MIA model of OA without affecting joint pathology, spinal glial cell activation or COX-2 expression in the PAG. SPHINX-31 did not reverse pain behaviour, but showed a potential effect on astrocyte activation and vascular-astrocyte association in the DL PAG relative VL PAG in the MIA model of OA.