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During normal vertebrate development, the ventricular zone (VZ) of the spinal cord reduces in size during the late prenatal period. In this process, termed dorsal obliteration, dorsal VZ progenitors move away from the VZ. Remaining ventral progenitors give rise to the ependymal zone of the adult central canal. At present, little is understood of the mechanisms that drive dorsal obliteration. Here I analyse dorsal obliteration in the prenatal mouse. I show that it occurs over a tight time-window, between E14- E17. Over this period, the dorsal midline is characterised by dorsal Nestin+ radial glial (dNRG) cells that display strong apical/endfeet expression of the apical polarity proteins CRB2 and ZO-1. Concomitantly, adjacent sub-dorsal VZ cells downregulate CRB2, ZO-1 and aPKC, and their nuclei become located away from the lumen. Ectopic transplantation of dNRG into the lumen of embryonic chick neural tube causes a disruption of progenitor patterning and the appearance of ectopic progenitor cells outside the neural tube, suggesting that dNRG cells secrete a diffusible factor that can disrupt neuroepithelial integrity. Previous bioinformatics studies had suggested the existence of an isoform of CRB2 that lacks the transmembrane and intercellular domain, and so may function as a secreted variant. Using a nested PCR approach, I find evidence that a isoform of CRB2 that lacks the transmembrane domain is specifically expressed in dNRG cells. Further, a protein encoded by this splice variant (secreted CRB2) can be secreted, and, when presented ectopically to chick neural tube, can disrupt neuroepithelial integrity. I therefore propose a model for dorsal obliteration in which secreted CRB2 can outcompete or interfere with a normal homodimerisation of transmembrane CRB2, leading to the destabilisation of the CRB2 complex and apical polarity, and the consequent delamination of subdorsal VZ cells. As a first step in testing this model, I develop an in vivo mouse slice culture assay in which to monitor cell behaviour during dorsal obliteration in real time. These studies provide evidence that the cell bodies of sub-dorsal cells immediately adjacent to elongating roof plate cells/dorsal Radial Glia migrate dorsally, using either the dorsal Radial Glia as a scaffold, or, potentially, re-orienting their own processes along the scaffold, and migrating along these. Finally, sub-dorsal cells appear to detach from the VZ in a process that may resemble apical abscission. Together, these studies suggest that a disruption of apical polarity proteins, driven through dorsal midline-secreted CRB2, is instrumental in dorsal obliteration. I discuss this model within the context of embryonic neurulation and discuss the implications for potential of the adult spinal cord.

It is well established that an observer will automatically follow the eye gaze of another person. Whether this automatic behaviour is influenced by the emotion displayed on the face has been a topic of intense interest yet previous research on healthy adults has failed to provide a consistent account. To address these inconsistencies, research has largely focused on examining the contribution of external factors and has ignored the relative, and perhaps critical, contribution of various internal factors. This ---thesis examines the contribution of a number of potentially important factors that might influence attention to the eye gaze of emotional facial expressions, with particular emphasis on biological and psychological internal factors. The first three experiments provide a baseline measure of gaze following of various facial expressions by healthy adults, and suggest that under standard conditions, and when a more ecologically valid target is included, gaze following is not influenced by the expression displayed on the face. Four further experiments highlight the importance of internal factors such as concurrent mood, the female menstrual cycle, and trait anxiety, in investigating emotional gaze following. Most significantly, it is apparent that in healthy adults, gaze following occurs more readily in positive contexts, and that this bias might be dysfunctional in individuals with high trait anxiety. These experiments provide an important insight into conditions under which rapid gaze following of emotional expressions proceeds. This thesis also serves to highlight the need for further gaze following research to take such individual differences into account.

A major goal in neurobiology is to study the mechanisms by which neurites navigate towards their targets to establish functional synapses. While a large amount of information is available on the extra cellular signals driving neuritogenesis and synaptogenesis, major gaps exist in the comprehension of the intracellular events. Rho family GTPases regulate the dynamic of the actin cytoskeleton and plays a crucial role in regulating neuronal development. In particular, Rac has been implicated in axon elongation and guidance, in dendritic growth and in the regulation of dendritic spines formation and plasticity. There are three Rac genes in mammals: Rac 1, Rac2 and Rac3, sharing about 90% protein identity, all participating in several functions, including actin dynamics and adhesion (Hall, 1998). Rac I is ubiquitous, while Rac2 is specific to the hematopoietic lineage and Rac3 is developmentally regulated 111 the vertebrate brain (Corbetta et aI., 2005), with the highest expressIOn during neurite branching and synaptogenesis (Bolis et aI., 2003). The co-expression of Racl and Rac3 in the vertebrate nervous system suggests that they are needed to solve individual developmental and maintenance issues in the nervous system. The aim of my work is to analyse the function of Rac I and Rac3 during nervous system development. I took advantage of both in vitro and in vivo approaches to answer this question. Comparison between wild-type and Rac3-/- hippocampal neurons showed no differences in neuritogenesis and synaptogenesis processes. On the other hand, Racl ~ depletion by RNAi in both wild-type and Rac3-/- hippocampal neurons during early stage of neuronal development showed specific alteration of the dendritic development without affecting axonal outgrowth. Reduced Racl levels impaired growth cone dynamics and Factin levels in both neurites and growth cones. These results show that endogenous Racl rather than Rac3 activity is essential for early dendritic development, but not required for the initial establishment of neuronal polarity and axonal development. Depletion of either VI Rac I or Rae3 during later stages of in vitro neuronal development did not severe dendritic spines morphogenesis, while double depletion strongly impaired the f01l11ation and maturation of dendritic spines. AlJ together these in vitro results reinforce the hypothesis that Rae I and Rae3 are both imp0l1ant for neuronal development underlying a specific role for Rac3 in later events of neuronal development. We next evaluated the effects of the depletion ofRacl and Rac3 in vivo. ] generated mice with conditional deletion of Racl in neurons (RacIN), by crossing Racl Flox/Flox mice (Walmsley et aI., 2003) with transgenic Synapsin-Cre mice (Zhu et aI., 2001) and double RacIN/Rac3-/- mice. As Rac3-/-, also Rae IN developed normally and was fertile. Both Rac3-/- and Racl N did not reveal major abn01l11alities in the brain cytoarchitecture, cell layering or alteration in the general organization of dendrites and synapses. Double RacIN/Rac3-/- mice died around 13 days after birth (P 13) and showed strong neurological defects. Morphological analysis revealed evident alterations in the hippocampus. The abnonnalities in the organization of the hippocampus may be correlated with the frequent spontaneous epileptic seizures developing after P7 in these mice. Vie found that double depletion of Racl and Rac3 induced an increase of WAVEl (Ser3IO) and PAKI (Thr423) phosphorylation, and reduced the total levels of RhoA. All together the data obtained in this thesis show for the first time that both RacI and Rac3 are important for neuronal development, while mutation of each gene leads to viable animals with no evident anatomical defects due to partial compensatory effects.

Microglia are the immune cell of the central nervous system and become reactive in response to infection or trauma. In Multiple Sclerosis (MS) microglia show early reactivity which may contribute to subsequent neurotoxicity. Glutamate has been implicated in the neurodegeneration in MS. Microglia express subtypes of metabotropic glutamate receptors (mGluRs), the activation of which can lead to microglial evoked neurotoxicity (group II) or neuroprotection (group III). This suggests that release of glutamate from neurones or glial cells may activate microglia via these receptors. Increased microglial reactivity during disease is possibly due to an imbalance of mGluR expression with neurotoxic group II increased above neuroprotective group III mGluRs. Microglial expression of mGluR subtypes was therefore investigated on activated compared with untreated cells. It was found that microglia exposed to myelin expressed increased levels of group II and mGluRl and 8 subtypes compared with control cells. MS is characterised by lesions in the CNS due to localised myelin breakdown. This research investigated the effects of microglial exposure to myelin and demonstrated that myelin induced microglial activation, upregulation of iNOS expression, and increased TNFa and nitrite release. Conditioned medium from these cells was toxic to neurones in culture. Microglia phagocytose myelin in vitro leading to cytokine release and increased affinity for oligodendrocytes, inducing their death by cell contact through TNF receptors. This study examined whether microglial phagocytosis could be modulated by stimulation of mGluRs and demonstrated that blocking group II or stimulation of group I or III mGluRs significantly reduced phagocytic activity. Since myelin induced toxicity was also reduced by blocking microglial group II or stimulating group III mGluRs, these results suggest that modulation of microglial mGluRs may be of therapeutic benefit in MS.

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