Activity-dependent neurotransmitter release is mediated by the Ca2+ -dependent fusion of synaptic vesicles at the active zone of the presynaptic plasma membrane and is vital for brain function. The process of vesicle priming, fusion and retrieval is precisely controlled and requires tight spatiotemporal coordination of multiple protein-protein interactions. This study shows that post-translational modification of the active zone protein Rab3 Interacting Molecule Ia (RIMIa) by Small Ubiquitinlike MOdifier I (SUMO-I) directs such interactions and is essential for fast synaptic vesicle exocytosis. Post-translational protein modification by SUMOylation is a fundamentally impOliant regulatory mechanism in nearly all cellular pathways with key roles in neuronal function including synapse formation and receptor trafficking. Presynaptically, SUMOylation has previously been shown to regulate neurotransmitter release, however the specific SUMO substrate proteins involved are unknown. Here, RIMIa is identified as a synaptic SUMO substrate where abrogation ofRIMIa SUMOylation at lysine residue 502 in neurons leads to severe defects in action potential-evoked presynaptic exocytosis and Ca2+ entry. SUMOylation of RIMIa promotes interactions with Ca2+ channel subunit Cav2.1 in a PDZ-dependent manner, with no apparent effects on vesicle docking or priming. FUlihermore, SUMO-l conjugation to RIMIa is required for the correct clustering of Cav2.1 and enhances the Ca2+ influx necessary for vesicular release. It is postulated that RIM I a exists as two populations with SUMOylation generating a 'molecular switch' whereby the unmodified form is responsible for vesicle docking and fusion events and upon SUMO-I conjugation, is responsible for Ca2+ channel clustering and mediating Ca2+ responses prior to release. This novel finding implicates SUMOylation as a regulator of synaptic vesicle exocytosis and more specifically, demonstrates that SUMOylation of RIMIa is a key determinant of rapid, synchronous neurotransmitter release. Results here provide further insight into the mechanisms underpinning synaptic function and dysfunction.

Cortical activity correlates of binary decision-making have been well-described in nonhuman primates (NHP), were cortical neurons display ramping activity that may reflect integration of evidence for choice alternatives. In rats, medial prefrontal cortex (mPFC) has been shown important for spatial decision-making tasks, when coordination with the hippocampus may enable integration of spatial information coded by place cells into frontal cortical processing. The hippocampus itself is implicated in decision-making and planning future actions by virtue of place cell sequences of non-local representations that can be predictive of future behaviour. During this phenomenon, the animal's head often orients towards and switches between potential options, as rats appear to consider the alternatives in a process described as vicarious trial and error (VTE). This thesis employs analyses of simultaneously recorded prelimbic cortex (PrL) and hippocampal population activity to characterise of this circuitry of spatial decision-making using a framework analogous to that used in NHP perceptual decision-making studies.

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This thesis reports work on synaptotagmin I, the isoform that occurs in adrenal chromaffin granules. The existence of a Ca<SUP>2+</SUP>-dependent conformational change in synaptotagmin was initially established by analysing the change in sensitivity of the protein to proteolytic digestion with trypsin in the presence and absence of Ca<SUP>2+</SUP>. An attempt was also made to investigate the Ca<SUP>2+</SUP>-binding activity of the protein with the lanthanide terbium (Tb<SUP>3+</SUP>), as a fluorescent probe for the Ca<SUP>2+</SUP>-binding sites but this approach proved to be of limited use, due to protein aggregation. Interaction of synaptotagmin with phospholipids was also found to be Ca<SUP>2+</SUP>-dependent and the protein was found to show selectivity for certain phospholipids. The interaction of the protein with calmodulin was tested using <I>in vitro</I> binding to immobilised calmodulin and also to calmodulin modified with a fluorescent dansyl group to act as a reporter. Both types of measurement indicated a Ca<SUP>2+</SUP>-dependent interaction between the proteins most probably due to affinity changes in calmodulin upon binding to Ca<SUP>2+</SUP>. Structural changes in synaptotagmin as a possible mechanism of action were investigated further using the biophysical techniques of circular dicheroism (CD) and light scattering. Changes in quaternary structural changes were not detectable by CD unless phospholipid vesicles were also mixed with the protein. Gel exclusion chromatography and native gel electrophoresis were used to support the data and indicated changes in quaternary structure. These techniques also suggested that the native protein, isolated from chromaffin granules, exists as a multimeric form. This study has demonstrated that synaptotagmin shows Ca<SUP>2+</SUP>-dependent interactions with calmodulin and phospholipids and also that it undergoes measurable structural changes in the presence and absence of Ca<SUP>2+</SUP>. This suggests that its action in exocytosis may be driven by a Ca<SUP>2+</SUP>-triggered conformational change.

Neural stem cells are the self-renewing and oligopotent cell population that generate constituent cell types of the nervous system. Cultured neural stem cells would offer researchers accessible opportunities to answer fundamental questions in both neurodevelopment and cell biology. Current strategies of maintaining neural stem/progenitor cells in vitro largely rely on neurosphere cultures (Reynolds and Weiss, 1992) and/or genetic immortalization (Frederiksen et al., 1988; Sah et al., 1997), These approaches raise concerns about cellular heterogeneity and potential cell transformation. Our lab has recently reported the establishment of adherent mouse Neural Stem (NS) cell lines that undergo symmetrical self-renewal without genetic immortalization (Conti et at., 2005; Pollard et al., 2006). Here, I apply this approach to human and rat foetal tissue and describe the derivation and characterization of human and rat NS cell lines. I established Human foetal NS cell lines from elective termination tissue. Human NS cells are propagated as stable cell lines in the presence of both epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), under which conditions they stably express neural precursor markers and exhibit negligible differentiation into neurons or glia. Human NS cells are induced to produce astrocytes, oligodendrocytes, and mature neurons upon exposure to appropriate differentiating conditions. Human NS cells are clonogenic stem cells. They are capable of generating clonal and tripotent cell lines from single deposited cells, demonstrating they represent self-renewing in vitro human neural stem cell populations. More importantly, human NS cells retain a diploid karyotype and constant neurogenic capacity for more than 100 generations, and their long-term stability does not require leukemia inhibitory factor (LW). Together with the demonstrations that human NS cells can be genetic modified and are accessible to multi-well time-lapse videomicroscopy, these cells creates the potential for high content genetic and chemical screens. In addition to human foetal tissue, adherent NS cells can also be derived from rat foetal brain and spinal cord. However, under standard expansion conditions supplemented with EGF and FGF2 (Conti et al. 2005), rat NS cells spontaneously become dormant after approximately 2 months expansion. Dormant rat cells exhibit stellate morphology and express the astroglial maker GFAP, but they still retain neural precursor makers such as Nestin and Sox2. I found that Bone Morphogenetic Protein (BMP) signals are responsible for generating quiescence of rat NS cells, and that FGF2 signaling inhibits BMP-induced astrocyte differentiation and therefore maintains stem cell potency. Applying NS cell conditioned medium or BMP antagonist Noggin could overcome cell quiescence, and by these means the long-term propagation of rat foetal NS cells can be maintained. In addition to foetal NS cells, Noggin also promotes the proliferation of adult rat subventricular zone (SVZ) neural precursors. These observations implies that the neurogenic but quiescent rat NS cells generated by BMP and FGF2 signals may reflect some characteristics of in vivo adult neural stem cells. Lastly, I undertake preliminary investigation of intracerebral transplantation using established NS cell lines. Mouse NS cells labelled with green fluorescent protein (GFP) were injected into cortex, striatum and hippocampus of both adult and neonatal mouse brain. I find NS cells can survive for at least 6 weeks after transplantation, although their migration appears limited. In adult brain, mouse NS cells differentiate into both astrocytes and morphological neurons expressing interneuron markers including Calretinin and Somatostatin. However, injected cells largely generate astrocyte in neonatal brain. These observations demonstrate that NS cells can be used as donor cells for transplantation studies. Future studies are required to evaluate how human and rat NS cell will behave after transplantation. It would also be informative to investigate whether cultured NS cells may contribute to functional repair in disease models.

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