The secretion of glucocorticoids from the adrenal gland is necessary for appropriate metabolism, immune regulation and responses to stress exposure. Glucocorticoid secretion occurs with defined patterns sensitive to physiological or pathological state, and communicates detailed messages to the cellular interior. These signals are conveyed in part by the genomic actions of the glucocorticoid receptor (GR), a ligand-activated transcription factor modifying the transcriptome by interacting with DNA. Little attention has been afforded to how GR-mediated genomic signalling interacts with the defined pattern of glucocorticoid secretion in vivo, or the varied physiological contexts in which glucocorticoid secretion occurs. In the first part of the thesis the dynamics of GR interaction with the pattern of hormone presentation is explored. Well recognised circadian glucocorticoid secretion comprises high amplitude pulses of adrenal secretory activity with an ultradian frequency (approximately one pulse/hr). The importance of the ultradian component of this secretion pattern, conserved through mammalian evolution has been previously suggested by detailed studies revealing an influence of physiological and pathophysiological state on rhythm components. By scrutiny of other systems possessing ultradian rhythms, we hypothesised a functional significance to the ultradian pattern of glucocorticoid release. The emerging hypothesis that ultradian secretion is important for appropriate gene regulation was examined. Utilising chromatin immunoprecipitation to define GR occupancy of endogenous gene regulatory regions, we describe a dynamic interaction of the GR with DNA binding sites in cell lines and animal tissues. GR responded individually to pulses of glucocorticoid exposure, and interacted with the genome in a manner defined temporally by ligand availability. The effect of this behaviour on the output of known glucocorticoid-regulated genes is discussed, incorporating the work of colleagues and collaborators using DNA binding ELISA and quantitative RT-PCR. The transcriptional program facilitated by this mode of interaction can be described as 'gene pulsing'. The second part of the thesis addresses context-dependence III glucocorticoid signalling .. Context -dependant behavioural and physiological responses to glucocorticoid likely anse through context-dependent molecular events. Access of transcription factors to DNA of higher organisms is controlled by the chromatin environment and by the availability of other transcription factors, which can either permit or hinder binding site accessibility. We hypothesised that the population of accessible binding sites for GR genome-wide is not absolute or fixed in any given cell type. Rather, changes in the chromatin landscape could arise from signalling components activated by stimulus exposure. Such changes may permit focused targeting of GR to DNA in a manner appropriate to the context of glucocorticoid exposure. Utilising the rodent response to stress as a context different from a baseline quiescent state, we designed experiments to test whether an equivalent elevation in plasma corticosterone produces the same genome-wide pattern of GR binding regardless of context (null hypothesis). Our approach depends upon high throughput sequencing technology to define regions of enriched GR binding determined by chromatin immunoprecipitation analysis (ChIP-seq). The thesis presents the experimental approach incorporating available preliminary data but does not address the hypothesis directly at this stage. Our results necessitate a revision of the classical view of glucocorticoid receptor function at the genome and hint at how the transcriptional program generated by GR's episodic association with the genome might be disrupted in human disease. A review of the clinical use of glucocorticoids, and the current approach to novel glucocorticoid therapeutics may be warranted.

Gap junctions (GJs) are intercellular channels connecting the cytoplasm of adjacent cells. This type of connection is an efficient way of cellular communications in many tissues including the central nervous system. Connexins are the proteins that constitute mammalian GJs, and Connexin43 (Cx43) is the most abundant isoform expressed in body cells. Cx43 has been detected within immature neural populations, but only in astrocytes in the adult brain and investigations have shown that Cx43 channel and adhesive properties largely influence neuronal differentiation of mouse neural progenitor (NP) cells. To date the role of Cx43 in neuronal differentiation remains unexplored in human systems, hence our study aimed to investigate the Cx43 participation in human NP differentiation. We largely detected Cx43 protein within the immature neural populations showing that protein expression occurred by fibroblast growth factor (FGF _2) stimulation through the ERKlj2 pathway; FGF _2 withdrawal induced NP differentiation and a progressive loss in Cx43 expression. Cx43's role in neuronal differentiation was explored by cloning lentiviral vectors (LV) coding for Cx43 or anti-Cx43 shRNA constructs and the protein knockdown resulted in an increase in neurons and a decrease in astrocytes, suggesting a role for Cx43 in human stem cell differentiation and neuronal fate. GJs mediate intercellular communications of several mouse and human embryonic stem (ES) cell lines; in our investigation we also showed the presence of functional GJ channels in human ES cells, as well as Cx43 protein expression. Manipulation of ES cells was attempted using LVs and results indicated that the CMV promoter in ES cells is largely inactive. In summary I demonstrated the active role of Cx43 in mechanisms that govern neurogenesis and differentiation of neural progenitor cells, furthermore we highlighted the importance of the internal promoter in LV constructs for genetic manipulation of embryonic ES cells.

NG2-glia, identified by their expression of the NG2 chondroitin sulphate proteoglycan (CSPG), represent a fifth glial cell population in the central nervous system (CNS). NG2-glia were originally considered to be oligodendrocyte progenitor cells (OPCs), but they have properties and distribution in the adult that is distinct from OPCs. NG2-glia have been shown to form contacts with axons at nodes of Ranvier in white matter and to respond to synaptically released glutamate in grey matter. Furthermore, NG2-glia are known to give rise to oligodendrocytes after demyelination in vivo and to form neurons and astrocytes in vitro. However, the function of NG2-glia remains largely undefined. Therefore, the aim of this thesis was to define the interrelations of NG2-glia with other elements in the CNS and test the hypothesis that they are multipotent neural stem cells (NSCs).

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Migraine is a common and disabling condition, affecting up to 15% of the population. This thesis sought to characterise certain aspects of the pharmacology of trigeminovascular nociceptive neurotransmission in neurons of the trigeminocervical complex (TCC) in the cat and also the rat ventroposteromedial thalamic nucleus (VPM). Electrical stimulation of the superior sagittal sinus (SSS) combined with the microiontophoretic ejection of L-glutamate were used to locate the cell bodies of neurons activated by trigeminovascular nociceptive input in the two regions. Agents were then microiontophoretically ejected onto these neurons to study their modulation of action potential firing. To investigate a potential role for the thalamus in migraine, commonly used antimigraine agents were microiontophoresed onto thalamocortical neurons within the VPM nucleus. Sodium valproate reversibly inhibited the response to SSS stimulation and L-glutamate ejection. The effects of GABA were also studied and found to have a similar action to valproate on trigeminovascular nociception. This appeared to involve activation of both GABAA and GABAB receptors. Naratriptan (a typical second generation triptan) similarly inhibited both responses. In addition to its action on serotonin 5-HT1B/1D receptors, this inhibition was also mediated by activation of 5- HT1B/1D receptors. Ergometrine maleate had a similar effect to naratriptan. Propranolol (a beta blocker) reversibly inhibited the response to SSS stimulation and L-glutamate ejection. This appeared to be through blockade of beta adrenoceptors as it could be antagonised by co-ejection of isoproterenol. The selective beta1 antagonist atenolol had a similar action to propranolol while beta2 and beta3 antagonists were ineffective. These results suggest that neurons in the VPM may be a target for anti-migraine medications and a fruitful subject for further research. The role of high threshold voltage gated calcium channels (VDCC) in trigeminovascular nociceptive neurotransmission by second order neurons of the TCC was also studied. The effects of selective peptide blockers of L-, N-, and P/Q-type VDCCs were examined. Microiontophoretic ejection of Agatoxin IVa/TK (P/Q-), co-conotoxin GVIa (L-) and calciseptine (N-type) reversibly inhibited neuronal firing in response to L-glutamate. This indicates an important post-synaptic role for VDCCs in regulating nociceptive neurotransmission within the TCC.

The VGF neurosecretory protein, first identified as a nerve growth factor (NGF) inducible gene product, is selectively synthesised predominantly in neuronal and neuroendocrine cells. The ~68 kDa VGF protein sequence is rich in paired basic amino acids, and thus the protein undergoes endoproteolytic cleavage to produce smaller peptides, which are stored in dense core vesicles and released upon stimulation via the regulated secretory pathway both in vitro and in vivo. Several of these VGF-derived peptides have been characterised and are involved in energy homeostasis, reproductive processes, synaptic plasticity as well as pain modulation. A number of studies have observed an increase in VGF gene expression in various pain models and more recently the VGF-derived peptides, TLQP-21, LQEQ-19 and TLQP-62 showed direct modulation of inflammatory and neuropathic pain when applied in vivo. The molecular mechanisms of action of VGF-derived peptides are not well understood and were investigated in this study. The TLQP-21 peptide, but not LQEQ-19, was shown to dose-dependently induce an increase in intracellular Ca2+ levels from cellular internal stores in brain- and spinal cord-derived primary microglia, in >65 % of the cell population in vitro. Three hour treatment of primary microglia with TLQP-21 (100 nM) induced a 2.78 fold increase in Ccl11 and a 2.28 fold decrease in Cxcl9 gene expression levels relative to the vehicle control (Student's t-test; p ≤ 0.05). Biochemical analysis using affinity chromatography and LC-MS/MS techniques identified the gC1q-R protein as a potential binding partner / receptor for TLQP-21. The gC1q-R protein is a ubiquitously expressed, multi-compartmental protein involved in complement activation, inflammatory processes and the plasma bradykinin formation pathway. These results tentatively suggest that TLQP-21 may contribute to the modulation of pain through activation of primary microglia and potentially involve interactions with components of the complement system. The findings highlight the importance of VGF-derived peptides in pain research and could lead to new perspectives and targets for pain therapeutics.

In the Ventrobasal (VB) thalamus, astrocytes are known to elicit NMDA-receptor mediated slow inward currents (SICs) spontaneously in neurons. Fluorescence imaging of astrocytes and patch clamp recordings from the thalamocortical (TC) neurons in the VB of 6-23 day old Wistar rats were performed. TC neurons exhibit spontaneous SICs at low frequencies (~0.0015Hz) that were inhibited by NMDA-receptor antagonists D-AP5 (50µM), and were insensitive to TTX (1µM) suggesting a non-neuronal origin. The effect of corticothalamic (CT) and sensory (Sen) afferent stimulation on astrocyte signalling was assessed by varying stimulus parameters. Moderate synaptic stimulation elicited astrocytic Ca2+ increases, but did not affect the incidence of spontaneous SICs. Prolonged synaptic stimulation induced a 265% increase in SIC frequency. This increase lasted over one hour after the cessation of synaptic stimulation, so revealing a Long Term Enhancement (LTE) of astrocyte-neuron signalling. LTE induction required group I mGluR activation. LTE SICs targeted NMDA-receptors located at extrasynaptic sites. LTE showed a developmental profile: from weeks 1-3, the SIC frequency was increased by an average 50%, 240% and 750% respectively. Prolonged exposure to glutamate (200µM) increased spontaneous SIC frequency by 1800%. This “chemical” form of LTE was prevented by the broad-spectrum excitatory amino acid transporter (EAAT) inhibitor TBOA (300µM) suggesting that glutamate uptake was a critical factor. My results therefore show complex glutamatergic signalling interactions between astrocytes and neurons. Furthermore, two previously unrecognised mechanisms of enhancing SIC frequency are described. The synaptically induced LTE represents a form of non-synaptic plasticity and a glial “memory” of previous synaptic activity whilst enhancement after prolonged glutamate exposure may represent a pathological glial signalling mechanism.

The noradrenaline reuptake transporter is located on the pre-synaptic membrane of noradrenic neurons. Its main function is to terminate the action of the neurotransmitter noradrenaline by reuptake back into the nerve terminal. Changes in the function and density of the noradrenaline reuptake transporter have been implicated in neurological disorders such as clinical depression and Alzheimer’s disease. In vivo imaging of the noradrenaline transporter using single photon emission computed tomography has been hampered by the lack of a suitable imaging agent. The information from imaging studies could lead to a better understanding of transporter function and the development of more efficient and faster acting drugs to treat the diseases associated it. For the first time, all four stereoisomers of an iodinated analogue of reboxetine were stereoselectively synthesised and biologically evaluated in an effort to understand the relationship between stereochemistry and potency. All four compounds were found to have nanomolar affinity for the noradrenaline transporter. Of most interest was the (2R,3S)-stereoisomer, which was identified as being as potent as the more studied (2S,3S)-stereoisomer. Therefore, a new series of iodoanalogues based on the (2R,3S)-stereochemical scaffold were synthesised and tested for their affinity with the noradrenaline transporter. This study revealed the derivative with ortho substitution on the phenoxy ring to be a potential lead for the development of a novel imaging agent for the noradrenaline transporter.