No description available.

Distinct neuronal populations show different sensltivity to global ischaemia, with hippocampal CAl neurons showing a greater vulnerability compared to cortical neurons. The oxygen/glucose deprivation (OGD) protocol was used in order to mimic the ischaemic insult in vitro. It has been previously demonstrated that OGD induces a rapid change in AMPA receptors (AMPARs) subunit composition at synapses in hippocampal neurons, promoting the expression of Ca2+-permeable AMPARs, responsible for the Ca2+ influx that triggers the damaging mechanisms that lead to neuronal death. In the CWTent study it is showed that the OGD-induced AMP AR trafficking mechanisms in cortical neurons differ from those in hippocampal neurons. This may contribute to the higher resistance in response to OGD displayed by cortical neurons. In order to explore the downstream consequences of these trafficking events, the small GTPases pathways were investigated. The OGD-induced activation of NMDARs and Ca2+_ penneable AMPARs stimulated the RacGEF Tiaml, via CaMKII activation in hippocampal neurons. However, OGD did not promote changes in Tiaml activation in cortical neurons. Differences in Tiaml activation affected the levels of GTP-bound active Racl , a small Rho GTPase involved in a wide variety of cellular processes, such as synapse and spine morphogenesis in neurons via actin remodelling. Rac l, under OGO conditions, significantly increased its activation in hippocampal neurons but in contrast, the activation decreased in cortical neurons. The differences discovered between hippocampal and cortical neurons potentially indicate an OGD-induced mechanism downstream of Ca +-permeable AMPARs that has repercussions on Racl activation that may affect spine density and morphology. This compromises neuron survival in response to OGD and could explain the differential vulnerability found in cortical and hippocampal neurons, as well as providing a new insight into neuroprotective mechanisms.

Group III metabotropic glutamate (mGlu) receptors (mGlu4/6/7/8) are presynaptic G-protein coupled receptors which act as auto- and hetero-receptors throughout the central nervous system. The development of mGlu8 receptor-selective agonist (S)-3,4-dicarboxyphenylglycine (DCPG), and antagonist (RS)-a-methyl-3,4- dicarboxyphenylglycine (MDCPG), in 2001 (Thomas et al., 2001), has led to a body of research investigating the possible function of this receptor subtype, and linking it to a number of pathological conditions including anxiety and epilepsy. The aim of the current work was to explore the role of mGlu8 within the hippocampal formation specifically, employing DCPG and MDCPG to assess its function in synaptic transmission and plasticity. The mGlu8-selectivity of these compounds was first assessed in the lateral perforant path (LPP) input to the dentate gyrus, a pathway known to express high levels of mGlu8 receptors. Field recordings in hippocampal slices from rat, and mGlu8 receptor knock-out (KO) mice revealed non-selective effects of DCPG at concentrations > 1 μM. Further experiments in slices from mGlu4, mGlu7 and mGlu2 KO mice, as well as a mGlu2-deficient substrain of Wistar rat, indicated that higher concentrations of the agonist activate the group II receptor subtype, mGlu2. Conversely, MDCPG appeared to be selective for mGlu8 at up to the highest concentration tested (30 μM). During this work in the LPP, DCPG was found to induce a novel form of mGlu8-mediated chemical long-term depression (LTD). This was induced by prolonged application of a high concentration (100 μM) of the agonist, and was more pronounced in older animals. Importantly, the LTD could be transiently reversed by application of MDCPG, up to two hours after washout of DCPG. Finally, patch-clamp recordings revealed inhibitory effects of DCPG on glutamatergic and GABAergic transmission onto subsets of CAl interneurons, but not pyramidal cells. This. is consistent with the known target-cell specific expression of mGlu8 within the hippocampus.

Pro-opiomelanocortin (POMC) is a precursor polypeptide that can be differentially cleaved into various biologically active peptides that have diverse effects. POMC is produced in neurones that are located in two distinct regions of the brain: the arcuate nucleus of the hypothalamus (ARC) and the nucleus of the solitary tract in the medulla (NTS). POMC neurones in the ARC have been well described in terms of their anatomical distribution and functional role, however NTS POMC neurones have not. The NTS is a major structure involved in cardiorespiratory regulation and is also linked to nociceptive processing, where NTS stimulation is antinociceptive. ~-endorphin, a cleavage peptide of POMe, is an opioid known to produce potent and lasting analgesia and can also affect cardiorespiratory function. NTS POMC neurones could provide a source of ~endorphin release within the brainstem. This project will test the hypothesis that NTS POMC neurones are involved in cardiorespiratory and nociceptive processing, via release of ~-endorphin. This project has employed viral vector-mediated strategies in POMC-Cre-ROSAGFP mice in order to: (i) opto-activate NTS POMC-Cre neurones and examine effects on cardiorespiratory control in the working heart-brainstem preparation; (ii) study the anatomical projections of NTS POMC-Cre neurones throughout the brain; and (iii) pharmacologically activate NTS POMC-Cre neurones in awake, behaving mice to examine influence on respiratory function, food intake and nociception. Opto-activation of NTS POMC-Cre neurones elicited profound cardiorespiratory responses, including a bradycardia, transient apnoea and increased respiratory sinus arrhythmia. These responses were attenuated by systemic naloxone. The bradycardias were mimicked by DAMGO microinjection into the nucleus ambiguus (NA), but not NTS. NTS POMC-Cre neuronal fibres projected to specific target sites within the brainstem, including the NA, ventral respiratory column, dorsal motor nucleus of the vagus, raphe obscurus nucleus, lateral reticular nucleus and hypoglossal nucleus. Pharmaco-activation of NTS POMCCre neurones resulted in increased tail-flick latencies, which were abolished by systemic naloxone. Taken together these results show that activation of NTS POMC-Cre neurones can produce antinociceptive effects, exert a potent facilitatory influence on cardiac vagal outflow and can inhibit the respiratory network. These responses are likely to be mediated by the opioid peptide ~-endorphin. The projections of POMC-Cre neuronal fibres outside of the NTS suggest that they form an output path to other CNS sites. These results suggest that NTS POMC-Cre neurones may have an opioid-mediated role in autonomic modulation and somatic nociception.

The periaqueductal grey matter (PAG) is a key structure in the expression of behavioural responses to pain and fear; however the motor pathways that mediate these crucial survival behaviours are poorly understood. Previous anatomical studies have suggested connections between the PAG and the cerebellum, a hindbrain motor structure which has recently also been implicated in emotional function and the expression of survival behaviours, such as freezing behaviour. This thesis provides evidence of a possible anatomical pathway between these two structures, and describes investigations into a possible function of this connection. In sodium pentobarbital anaesthetised rats, cerebellar cortical field potentia Is were recorded in response to electrical stimulation in the PAG. Stimulation at both "IPAG and dlPAG sites was found to evoke bilateral climbing fibre field potentia Is in lateral regions of cerebellar vermallobule VIII. Fos expression was significantly increased in the fastigial nucleus A module in animals that underwent stereotaxic procedures, compared to anaesthetic control animals. Animals that received noxious pinches of the snout also tended to display greater numbers of Fos labelled neurons in this region. Animals that underwent excitation of the vlPAG with dl-homocysteic acid (DLH) displayed significantly fewer Fos labelled neurons in the A module than saline injected animals. This may reflect a vlPAG mediated depression of the noxious input to the cerebellum originating from stereotaxic and surgical procedures employed during the experiment. The functional significance of the PAG-Iobule VIII connection was investigated by lesioning connections to/from lateral lobule VIII using CTb-saporin. On average, lesioned animals displayed significantly reduced freezing behaviour during presentation of an aversively conditioned auditory tone. The animals that displayed the greatest reduction in freezing also displayed significantly fewer Fos labelled neurons in the caudal vIPAG. This suggests that vermallobule VIII supports neuronal activation of the caudal vlPAG during exposure to conditioned fear stimuli.

Recent evidence from studies using cognitive neuropsychological testing in human depression suggests that specific cognitive mechanisms play an important role in the development and perpetuation of depressive illness. These studies suggest that quantifying objective measures of affective state and affective processing may provide a new opportunity for developing translational animal models. This thesis describes the validation of a novel behavioural approach, the affective bias test CABT) designed to facilitate the investigation of cognitive affective processing biases in rodents. Pharmacological and psychosocial anti- and prodepressant treatments were shown to induce affective biases that are predictive of their effects in man. These effects were shown to increase with successive experiences encountered during affective treatment, and the data suggest that the treatments may act by modifying the consolidation of affective memory. The NMDA antagonist ketamine was observed to attenuate acquired negative affective biases via disruption of neurotransmission in the prefrontal cortex, while the monoamine reuptake inhibitor venlafaxine positively biased new learning through actions in the amygdala. This pattern of results suggests that the neuropsychological mechanisms of delayed- and rapid-onset antidepressants are neurally and temporally dissociated. A pilot study was also conducted to investigate the hypothesis that negative affective biases induced by prolonged negative affective state generalise to behaviours associated with social and exploratory behaviour, and this study has provided a methodological basis for future studies investigating the role of chronic stress in depression. Together the work presented in this thesis supports a cognitive neuropsychological mechanism for the development and treatment of depression and demonstrates face, predictive and construct validity for the ABT as a preclinical assay for use in drug safety and development.

No description available.

Studies have been performed to detail the phenomenology, investigate the skeletal changes and explore the spinal biomechanics underlying the main axial deformities – Pisa syndrome and camptocormia in Parkinson’s disease. Results demonstrate that the clinical picture of these deformities varies greatly but that certain particular features allow distinction from other neurological, muscular and bony aetiologies. The tone of the axial muscles, the level at which spinal flexion occurs, the patient’s ability and method to try to overcome the chronically abnormal posture, and the flexibility or fixity of the trunk provide clinical pointers to the likely underlying cause. The scoliotic curve in a patient with Pisa syndrome was C-shaped, involved a large element of collapse and occurred without evidence of a secondary upper compensatory curvature (S-shaped curve). On supine imaging patients with camptocormia were severely mechanically disadvantaged as a result of their alordotic lumbar spines in relation to pelvic angulation. This lumbar alordosis may reflect the effects of Parkinson’s disease on the axial musculature, particularly in those with axial akinetic rigid predominant PD. Radiological examination also demonstrated that Pisa syndrome was different from de novo degenerative scoliosis and camptocormia not typical of adult onset degenerative kyphosis. Fixed bony changes were rare but the severity of these postural deformities and their consequent effects (e.g. knee flexion contractures, gluteal muscle atrophy) are likely to render conservative interventions unsuccessful unless instigated very early in the evolution of the abnormal posture.

Parkinson’s disease (PD) is an incurable neurodegenerative disease. Although the majority of PD cases are sporadic, 5-10% of cases are inherited. Studies of sporadic and genetic forms of PD suggest shared pathogenesis such as mitochondrial dysfunction. Mutations in the gene encoding Parkin are the most common cause of autosomal recessive, young-onset PD. Parkin has been shown to regulate mitochondrial quality control (mitophagy), however the molecular pathways that regulate Parkin activity remain poorly characterised. MEKK3/p38, MAPK/ERK, and PI3K/Akt signalling pathways have been described in association with Parkin regulation. In this thesis, I have investigated whether activation of any of these pathways could lead to Parkin phosphorylation by utilising inducible cell lines overexpressing MEKK3-ER, Raf¬ER or Akt-ER genes. I found that Parkin was not phosphorylated following the activation of the p38, ERK and Akt pathways. In an attempt to depolarise mitochondria in neuroblastoma SH-SY5Y cells lines by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), I found that Parkin was phosphorylated at serine 101 (S101). In order to investigate the role of phosphorylation of Parkin S101 in mitochondrial quality control, I established SH-SY5Y clones stably expressing wild type (WT), non-phosphorylatable (S101A), or phosphomimetic (S101D) FLAG-Parkin. I found that this phosphorylation is associated with increased Parkin’s E3 ligase activity. S101A cells showed deficiencies in translocation of Parkin to depolarised mitochondria, ubiquitination of outer mitochondrial membrane proteins, p62 (an autophagy adaptor) recruitment, perinuclear mitochondrial clustering and mitophagy. Overall the work presented in this thesis demonstrates that Parkin is activated during mitochondrial depolarisation, and that the regulation of Parkin function via phosphorylation at S101 plays an important role in mitochondrial quality control associated with PD pathophysiology.

The neural tube is the developmental precursor of the central nervous system (CNS). Neural tube defects (NTDs) are among the commonest birth defects, affecting approximately 1 in 1000 pregnancies. They occur when the neural tube fails to close completely during neurulation, and result in an open region of the CNS. Spina bifida is an NTD affecting the spinal region, and it can lead to lifelong disability, including incontinence, motor difficulties, and paralysis below the level of the lesion. Exencephaly is an NTD affecting the cranial region, and is not compatible with life. Splotch mice carry a mutation in the Pax3 gene which leads to a functionally null Pax3 protein. Pax3 is important in the development of a number of tissues, and mutant embryos have defects in muscle development, and neural crest-derived tissues, such as the heart, peripheral nervous system and melanocytes. Additionally, embryos develop NTDs; they demonstrate spina bifida with complete penetrance and exencephaly with partial penetrance. The cellular mechanism behind the development of NTDs in Splotch embryos is not well understood. However excess apoptosis, premature neuronal differentiation, and reduced proliferation in the neural tube have all been proposed as potential causes. Furthermore, research has suggested a potential link between Pax3 and canonical Wnt signalling. The aim of this research was to study cellular defects in Splotch mice which are potentially causative of NTDs, and also to study the interaction between Pax3 and canonical Wnt signalling. It was found that excess apoptosis and premature neuronal differentiation are not causative of spina bifida in Splotch mice. However, reduced proliferation is present in the neural tube, and may be causative. Additionally, β-catenin loss- and gain-of-function mutations were used to study interaction between Pax3 and canonical Wnt signalling. β-catenin loss-of-function reduces Pax3 expression, and β-catenin gain-of-function worsens NTDs in Pax3 mutant embryos, whereas loss-of-function partially rescues exencephaly, but not spina bifida, in these embryos. β-catenin gain- and loss-of-function both also worsen neural crest defects in Pax3 mutant embryos. Therefore, Canonical Wnt signalling interacts with Pax3 during development, and affects the cranial and spinal regions of the neural tube differently. In summary, Pax3 mutation results in a number of defects in developing embryos, including NTDs and neural crest defects. Cellular processes have been studied in this research to identify abnormalities which could be causative of these defects, and a potential molecular link between Pax3 and the canonical Wnt signalling pathway has been identified, which could contribute to the observed phenotype.