PrP undergoes three major post-translational changes, N-terminal cleavage, N-linked glycosylation and the cleavage of a C-terminal peptide with the subsequent addition of a phosphatidylinositolglycolpipid (GPI-anchor), which is responsible for the protein's attachment to the membrane (ref, Stahl 1987). Most PrP<SUP>sc</SUP> appears to have an intact GPI-anchor although it cannot be released from tissue culture cells by the action of PIPLC as the wild-type can (Stahl 1990). However, 15% of PrP<SUP>sc</SUP> from hamster brain has been shown to be truncated at amino acid 228, these proteins having no GPI-anchor (Stahl 1990). The significance of these truncated proteins has not yet been elucidated. It is possible that the presence of a small amount of an abnormal, truncated protein could act as a seed for a conformational change that would result in the conversion of PrP<SUP>c</SUP> to PrP<SUP>sc</SUP> (Prusiner 1991). This project sets out to investigate the differences between the cellular processing of the wild type GPI-anchored protein and a mutant protein where addition of the GPI has been prevented. The mutant is investigated to show that the engineered mutation does indeed give rise to a GPI-less form and the size, glycosylation status, immunoreactivity and cellular location of the two proteins are investigated firstly in a cell-free system and secondly in tissue culture cells. The question of whether PrP<SUP>sc</SUP> can be produced when the GPI-anchor is absent will be addressed by subsequent studies of these mutations in transgenic mice.

Spinal nociceptive processing undergoes extensive maturation in the postnatal period. Dorsal hom neurons are more easily excited by peripheral stimuli in young animals and nociceptive reflexes are less well directed. Since circuits in the central nervous system require a balance of excitation and inhibition, I hypothesised that the maturation of inhibitory circuitry is responsible for changing nociceptive processing in postnatal animals. To test this I have investigated the maturation of segmental and descending inhibition in the anaesthetised and decerebrate rat dorsal hom using in vivo electrophysiological methods. First, I examined the developmental regulation of inhibitory signalling in spinal nociceptive circuits. Single cells in the lumbar dorsal horn, characterised by their responses to hindpaw cutaneous stimulation, were recorded at two postnatal ages over a prolonged period in the presence and absence of a GABAAR antagonist. The results show that at the circuit level, GABAergic signalling does not change between postnatal days 3 and 21 and that blocking GABA activity is equally excitatory at both ages. Next, I examined the role of descending activity from supraspinal centres upon single dorsal hom cell activity at different ages. The results show that while descending inhibition of spinal nociceptive activity is present in young animals, there is also an excitatory descending influence upon young dorsal hom cells that is not observed in older animals. Finally, I mapped the spatial pattern of inhibitory receptive fields at different ages. The results show that contralateral inhibitory fields are less spatially restricted in young animals and are activated by both low and high intensity stimulation. A model is proposed to show how inhibitory receptive field organisation may determine higher-level sensory processing. In conclusion, the postnatal maturation of spinal nociceptive processing is likely due to maturation of local and descending inhibitory activity within spinal circuits.

In this work, I have characterised the biological functions of Kidins220 (Kinase D interacting substrate of 220 kDa) in both an in vitro and in vivo context. Kidins220 is a conserved membrane protein mainly expressed in neuronal cells, which has been implicated in the process of neuronal differentiation in response to neurotrophic stimuli. In the first part of this study, I present an analysis of the molecular mechanism regulating the intracellular trafficking of Kidins220 in a rat pheochromocytoma cell line (PC 12), which upon treatment with nerve growth factor (NGF) differentiates into neuronal-like cells resembling sympathetic neurons. Kidins220 transport to neurite tips is driven by the microtubule-dependent motor complex kinesin-1. Perturbation of Kidins220 trafficking in this cellular system reduces the activation of the signalling cascades initiated by NGF and impairs neuronal differentiation. I was interested in understanding the mechanisms that might modulate the association of Kidins220 to kinesin-1. Kidins220 recruits kinesin-1 via a novel binding motif, which does not share any similarities with the known kinesin-1- interacting signatures. I found that Abelson tyrosine kinase (Abl) phosphorylates Kidins220 at the level of this kinesin interacting motif, thus inhibiting the binding to kinesin light chain. These preliminary results suggest that phosphorylation might act as a molecular switch, to mediate the release of Kidins220 from the kinesin-1 motor complex. In the second part of this work, I analysed the effects of Kidins220 depletion in living organisms. I tackled this problem by targeting Kidins220 by RNA interference in Drosophila melanogaster. In an independent approach, I have generated a construct, based on the Cre/LoxP recombination system, for the conditional knockout of the Kidins220 gene in mice. Kidins220 null animals die at late stages of embryonic development, and display severe cardiovascular and neurological defects. Their phenotype is described in detail using a variety of immunohistochemical and cell biological approaches. This work therefore presents new evidence supporting the notion that Kidins220 plays an important role in regulating not only neuronal function and differentiation, but also other key developmental processes such as cardiac development.

There is a need to improve our understanding of the mechanisms of pain, especially neuropathic pain in order to develop new analgesic drugs based on opioids. In recent years, the cloning of the novel opioid receptor like-1 (ORLi/NOP) receptor and studies on the effectiveness of opioids in pain models provides a basis for potential novel therapy. This thesis is based on nociceptin/orphanin FQ and its receptor ORLi, which represent a novel peptide/receptor system pharmacologically different from classical opioid systems. Nociceptin regulates several biological functions, both at the peripheral and central levels therefore, the ORLi receptor may be viewed as a novel target for drug development. However, the pharmacology of this receptor is still under study, with few molecules selectively acting on this receptor. Little is known about the physiological roles of this new opioid system. Using an in vivo electrophysiology study, spinal effects of nociceptin were investigated on deep dorsal horn neurones in normal, sham operated and neuropathic rats. Nociceptin induced a greater dose-dependent inhibition in normal animals when compared with the neuropathic and sham operated animals which were the least inhibited. Additionally to clarify the role of nociceptin and its receptor in the spinal processing of pain a non peptide antagonist and agonist of nociceptin were studied. Another objective of this thesis was to study the interaction between nociceptin and cholecystokinin (CCK), an anti-opioid peptide. CCK enhanced the inhibitory effect of nociceptin in sham operated and neuropathic animals, whereas in normal animals CCK had the expected antiopioid action. Furthermore, this thesis emphasizes the importance of opioid receptors located on lamina I expressing NK1 receptors in the modulation of spinal analgesia of nociceptin when compared to D-Pen2, D-Pen5 enkephalin (DPDPE), a delta-opioid agonist. Finally, this thesis suggests a potential therapeutic value of oxytocin in the treatment of neuropathic pain.

No description available.

Mole-rats (of the Bathyergidae family) provide a unique taxonomic group for studying many aspects of neurobiology. In this family, ecological constraints have led to diverse social and reproductive strategies. Eusociality occurs in two species of Bathyergidae rodents; naked mole-rats (Heterocephalus glaber) and Damaraland mole-rats (Fukomys damarensis). The females of these two eusocial species are at the extreme end of the socially-induced infertility continuum whereby ovulation is physiologically blocked. This family provides an ideal model to glean insight into the neurobiological mechanisms of reproductive suppression, eusocial behaviour and neuroplasticity. In this thesis, I report that: (1) naked mole-rats display substantial numbers of kisspeptin-immunoreactive cell bodies in the rostral periventricular region of the third ventricle, paraventricular hypothalamic nucleus, arcuate nucleus and dorsomedial nucleus, irrespective of reproductive state, sex or presence of gonads, (2) reproductive naked mole-rats display significantly higher number of kisspeptin-immunoreactive cell bodies in the rostral periventricular region of the third ventricle and paraventricular hypothalamic nucleus than subordinates, (3) subordinate Damaraland mole-rats have an absence of a RFamide neuronal population that is found in abundance in reproductive Damaraland mole-rats, with no effect of gonadectomy or sex differences, (4) the presence of a sexually monomorphic population of hypothalamic tyrosine hydroxylase neurones indicates the lack of sexual differentiation in the brains of naked mole-rats, (5) the shared occurrence of oxytocin and its receptor in the nucleus accumbens of both eusocial naked mole-rats and eusocial Damaraland mole-rats indicates the possibility of convergent evolution in this family, (6) two novel locations for vasopressin receptor binding in the brains of Damaraland mole-rats have evolved independently during the divergence of this species from a common ancestor, and (7) naked mole-rats have a remarkably low rate of adult hippocampal neurogenesis, a low rate that is shared with other long-lived, group-living animals.

Exposure to an acutely stressful event leads to activation of NMDA receptors, initiating a cascade of MAPK/ERK signalling, epigenetic modifications and lEG induction in sparse neurons of the dentate gyrus (DG); these changes have been shown to be vitally important for long-term memory formation. The glucocorticoid receptor also plays an important role in this cascade. The work presented in this thesis continues to explore this signalling pathway in the DG and CA regions of the hippocampus in the context of Morris water maze (MWM) learning. Phosphorylation of ERK1/2, phosphorylation of histone H3 at serine 10 and NMDAR-dependent induction of immediate early genes (lEGs) were found to occur in sparse neurons of the hippocampus following MWM training. Although these lEGs are known to be important for learning and memory, the epigenetic and gene induction changes were induced in both MWM-trained and swim control (sq rats to a remarkably similar extent. Nevertheless, a significantly higher Arc induction was found in the MWM group in the CA regions on the second day of training. Chromatin immuno-precipitation was used to investigate the epigenetic mark H3K9acSlOp, which was found to be associated with lEG promoters both under baseline conditions and after MWM or SC procedures; further study with an increased sample number will be required to confirm whether there is increased association after training. Finally, RNA-sequencing was used to identify late-response genes up-regulated in the DG three hours after MWM and SC procedures. Many genes were identified which are regulated by MWM or SC training; however, there were no genes differentially regulated between the MWM and SC groups. This thesis has advanced our knowledge of the molecular and gene expression changes occurring within neurons of different hippocampal sub-regions after learning under stressful conditions. In addition, it has cast light on the factors determining lEG responses in hippocampal neurons and has led to the identification of novel genes previously not known to be regulated after MWM learning.

After a brief review of the history and philosophy of neural modelling, several attractor neural networks are studied in some detail. Firstly a variation of the Hopfield Model employed to perform symmetry invariant pattern recognition is considered. It is shown that parallel dynamics tend to perform a symmetry-transformationof the network configuration at each update. In contrast serial dynamics tend to drive the network configuration into a symmetry invariant. The component of the interactions that drive the aforementioned dynamic tendencies act as a noise upon the Hopfield interactions. However replica symmetric theory shows that an extensive number of patterns may be stored whilst allowing symmetry invariant pattern recognition. The performance of Gardner optimal interactions, that optimise the performance of a perceptron, is examined in the context of attractor neural networks. The discussion is restricted to randomly dilute networks for which dynamical equations for the overlaps are available. A general analysis of these equations is performed and the transitions to no memory categorised. In particular the conditions for a point, tricritical in nature, to exist in the α<i>T</i>- plane are derived. Retrieval phase diagrams for the optimal interactions with and without errors in storage are constructed. The case of sparse spatial coding is then investigated by considering two connection rules, Covariance and Willshaw, that have the storage capacities of the form of the Gardner optimal connections as the bias of the patterns becomes very large. In both cases the choice of threshold is crucial in order to achieve maximum storage, and also controls the basins of attraction of the memories. Both connection schemes exhibit an undesirable high activity attractor, but in the Willshaw case this may be suppressed by introducing an activity dependent inhibition. In order to bring network models into close contact with biological experiment, the problem of firing rates is discussed. A model is then proposed that uses a biologically realistic dynamics and incorporates a variety of other biological features. Graphic displays from computer simulation of the model are presented and associative retrieval can be seen to occur whilst the network functions in a manner that is reminiscent of the results of biological experiments.

The influence of prenatal exposure to aluminium sulphate (Al<SUB>2</SUB>(SO<SUB>4</SUB>)<SUB>3</SUB>) on the behavioural development of mice (<i>Mus musculus</i>) from two inbred strains was examined. Pregnant CBA/T6 and C57BL/6J mice were exposed by intraperitoneal (i.p.) injection (200mg/kg) or orally (750, 1000 and 1250mg/L) to Al<SUB>2</SUB>(SO<SUB>4</SUB>)<SUB>3</SUB>. On postnatal day one, pups were cross-fostered and tested in a variety of ethological measures, from birth to adulthood, to assess effects on the mother and the behavioural development of the pups. As a neurochemical marker for the cholinergic system, choline acetyltransferase (ChAT) activity was measured at different developmental stages. Breeding performance, the length of gestation and sex ratio were unaffected by Al exposure administered via the i.p. route. There was a transient reduction in maternal weight gain during gestation. CBA pups born to Al-treated mothers exhibited lower body weights at birth; this reduction persisted into adulthood only in treated pups reared by treated mothers and was more pronounced in the case of female mice. The body weight changes were accompanied by delays in the maturation of several of the tests of sensory-motor development. Al-treated CBA females were hypoactive at weaning compared to controls, whilst the converse was true for males. 77% control and 55% treated males reached criterion in a maze test and controls required fewer days to do so. The effect of Al exposure on the cholinergic system was dependent on the region of the brain studied, and still showed significant effects in the adult. <i>In utero</i> exposure to injected Al resulted in a reduction in the rate of ultrasonic calling by CBA pups and was accompanied by a delay in the timing of peak calling. C57 pups were not affected to the same degree. Exposure to oral Al caused a similar but less obvious trend towards a diminished calling. The inclusion of the recording of ultrasonic calling is recommended in any test battery aimed at assessing behavioural teratogenicity.

The exact mechanism by which the hippocampus contributes to memory formation, and the underlying molecular and biochemical processes are not understood. One approach to understanding hippocampal function is through transgenesis. The identification of genes specifically expressed in this formation would provide promoters capable of directly reporter gene expression to the hippocampus. This thesis aims to identify genes expressed in the hippocampus. Such genes would permit targeting hippocampus-specific gene expression, while their identity and function within the hippocampus may also be characterized. Differential hybridization techniques, employing cDNA libraries prepared from hippocampus or other brain regions, have been used in an attempt to identify genes expressed either specifically or predominantly in the hippocampus. One cDNA clone isolated, Hct1 (hippocampal transcript), is further characterized. The expression pattern of Hct1, by Northern analysis and <I>in situ</I> hybridization, shows significantly enriched expression within the adult rat hippocampus, as well as weak expression in the liver. Sequencing of cDNA and genomic clones of Hct1 identified the encoded protein as a novel member of the cytochrome P-450 superfamily, a large group of enzymes involved in the oxidative metabolism of a variety of steroids, fatty acids and xenobiotics. Hct1 may represent a new family of cytochrome P-450 genes. The possible role of this enzyme in hippocampal function is discussed.