Fbw7 belongs to the family of F-box proteins, which function as substrate recognition subunits of SCF complexes. Fbw7 controls the stability of several proteins including cyclin E, the Notch intracellular domain and c-Myc. In 2004 our lab additionally identified phospho-c-Jun as an Fbw7 substrate, c-Jun is part of the AP-1 transcription complex, whose activity is strongly induced in response to numerous signals such as growth factors, cytokines and extracellular stresses. Furthermore elevated phospho-c-Jun levels induce neuronal apoptosis. To investigate the significance of c-Jun regulation by Fbw7 in the nervous system, I generated mice harbouring a floxed fbw7 allele, fbw7. fbw7 mice were bred to various Cre transgenic lines that express the Cre recombinase under nervous system specific promoters to obtain mice with a tissue specific deletion of Fbw7. I confirmed published results that ubiquitous deletion of Fbw7 mediated by PGK-Cre is lethal. To delete Fbw7 at the stage of neuronal precursors, fbwff mice were crossed to the Nestin-cre line (fbw7). These mice die perinatally and show an increase in apoptosis at El6. As the lethality of the fbw7AN mice does not allow the investigation of Fbw7 in the adult nervous system, further crosses, using other cre-transgenic lines, were set up. Fbw7 deletion in postmitotic neurons (fbw7ApN) causes a Parkinson's disease like phenotype with a severe hindlimb tremor and a reduced cortical cellularity. Fbw7 deletion in the cerebellar vermis (fbw7ACb) resulted in cerebella that are characterised by a reduced size, foliation defects accompanied by an astrocytic gliosis and a phospho-c-Jun dependent Purkinje cell loss. Concomitant deletion of c-Jun in the cerebellum (fbw7ACb :c-junACb) partially rescues the cerebellar phenotype caused by Fbw7 deletion. Thus the data in this thesis demonstrate a role for Fbw7 in cerebellar development and the central nervous system and identify c-Jun as an essential Fbw7 substrate in the nervous system.

The hippocampal formation is known to support several different types of spatial representation, and to play an important role in detecting environmental novelty. A striking aspect of hippocampal physiology is the theta rhythm, recorded in the electroencephalogram (EEG). This thesis investigates the role of the theta rhythm as an organising principle of the hippocampal formation, and in particular its role in the detection of novelty. In the first experimental chapter, the behavioural correlates of principal cells across several regions of the hippocampal formation - head direction and conjunctive place by direction cells in the presubiculum, place cells in CA1 and grid cells in the MEC - are studied, with an emphasis on the contribution of running speed. Subpopulations of cells, both positively and a minority which are negatively modulated by speed are found in each region. The second experimental chapter investigates the predictions of a recent model of entorhinal cortical grid cells. The model, based on the interference between somatic and dendritic oscillators, predicts that intrinsic firing frequency should exceed EEG theta frequency by a greater amount for small grids than for large grids and by a greater amount during fast running compared to slow. These relationships are confirmed in electrophysiological recordings in freely-moving rats. The third experimental chapter reports the results of a detailed examination of the rodent EEG under conditions of novelty and familiarity. The oscillatory interference model predicts a reduction in the theta frequency in a novel environmental context. This is substantiated by the data and reveals a new mechanism for signalling novelty in the brain. The fourth experimental chapter probes the specific roles played by the subiculum and CA1. There is debate as to whether the subiculum is an input as well as, traditionally considered, an output of the HF. I propose that subiculum is capable of informing CA1 and data is presented to show that subicular firing occurs earlier in the theta cycle and anticipates position further ahead than firing in CA1 does, and that this difference is modulated by familiarity with the environment.

This study aimed at providing a clearer understanding of the functional significance of specific receptor subtypes in inhibitory and excitatory synaptic transmission in cerebellar granule cells. These cells have a simple morphology, typically consisting of a small soma with four short dendrites. Each receives a single excitatory input, which activates both AMPA and NMDA receptors. Granule cells also receive GABA-mediated inhibitory input from several Golgi cells. I have used mainly patch-clamp recording methods combined with use of pharmacological tools. Various strategies (examination of developmental changes, and use of transgenic animals) have been used to provide information about the importance of specific NMDA-, AMPA' and GABM receptor subunits in the transmission process. I examined developmental changes (~P8 to P60) in the contribution of NMDARs to mossy-fibre-to granule cell (MF-GC) synaptic currents in young and mature mice. Initial data suggested that NMDAR-mediated component was absent from quantal events recorded at mature synapses in these cells. I have used a number of different approaches to determine whether this can be explained by a lack of synaptic MDARS at the mature synapse. We investigated the importance of the two glutamate receptor subunits that are crucial to excitatory transmission in cerebellar granule cells - namely NR1, and GluR4. We used NR1 lox and GluR4lox mice, respectively, for these experiments. The data suggest that deletion of GluR4 may produce a compensatory change, consistent with the view that GluR4-containing AMPARS are important for transmission at the MF-GC synapse. Deletion of NR1 caused no compensatory change. We investigated a GABAA receptor subunit that is thought to be inhibitory transmission in these cells. Knockout of the gamma-2 subunit, caused a loss of IPSCs in these cells. However, there was no compensatory increase in tonic conductance. Furthermore, there was no compensatory change in intrinsic membrane properties in these cells. We also found no compensatory change in Purkinje cell firing. Thus any compensation, if present, must arise before the final output by Purkinje cells is completed.

Neural tube defects (NTDs), such as exencephaly and spina bifida, occur when the neural tube fails to close properly during embryonic development. Folic acid supplementation has been shown to effectively reduce the occurrence of NTDs and low folate is a known risk factor for human NTDs. However despite extensive research, the mechanism explaining how the folate status affects the incidence of NTDs remains unknown. One effect of sub-optimal folate could be suppression of the methylation cycle, which is interlinked with the folate cycle. To explore this hypothesis, a method has been developed to quantitatively measure the substrate, S-Adenosylmethionine (SAM), and the product, S-Adenosylhomocysteine (SAH), of methylation reactions in neurulation-stage mouse embryos by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). To investigate the link between methylation perturbation and neural tube defects, the abundance of SAM and SAH has been quantified in a series of experimental models and human NTD cell lines. Using a folate-deficient diet, this study also investigates folate deficiency using mouse models (splotch and curly tail) for NTDs. Results illustrate that folate deficiency, in both mouse models, caused an increase in the incidence of neural tube defects and, embryos were growth retarded. To examine the effect the folate deficient diet was having on endogenous folate metabolism, embryonic folate levels were analysed, in addition to maternal folate. Furthermore, using the established Ic-ms/ms method, the effect of folate deficiency on the abundance of SAM and SAH was determined. Increasing evidence suggests that myo-inositol may prove effective in preventing NTDs, in conjunction with folic acid. Thus, a double-blind, randomised clinical trial has been designed to investigate this and, in order to monitor patient compliance an LC-MS/MS method has been developed to quantify myo-inositol in urine.

Influential theories of emotion propose that afferent bodily information is central to the experience of emotional feeling states. Neuroanatomical studies suggest a phylogenetically unique afferent neural system in primates that conveys information regarding motivationally salient aspects of the physiological condition of the viscera and other bodily organs and supports a representation of homeostatic afferent activity engendering sensations including nausea, pain and muscular aches. This thesis focuses on brain-body interactions in neural signalling of sickness and communication of emotional distress. I begin by presenting investigations into the role of visceral afferents travelling with the autonomic nerves in the central communication of bodily inflammation and the mechanisms underlying cognitive, emotional and motivational components of sickness. Using a model of inflammation (Typhoid vaccination) and functional magnetic resonance imaging (fMRl) I show that peripheral inflammation activates the hierarchy of brain regions encoding a representation of bodily state consistent with an autonomic afferent mechanism, wherein differential insula, subgenual cingulate and substantia nigra activity predict fatigue, mood change and psychomotor retardation respectively. I then investigate the role of organ specific autonomic signals in the differential responses to bloody (mutilation) and core-ingestive forms of disgust. I present fMRl, heart rate and electrogastrogram data which suggest organ-specificity in bodily responses to different forms of disgust related to these distinct peripheral channels. Finally I present data investigating the role of autonomic signals in the communication of distress and demonstrate that pupillary signals selectively modulate perception of another’s sadness. Using fMRl and pupillometry I show that another’s pupillary signals in the context of sadness modulate activity within the observer’s brain in face processing regions. Further in sadness observed pupil size leads to a contagious mirroring of the observer’s own pupil size via a mechanism that recruits brainstem pupillary control (Edinger Westphal) nuclei.

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