The function and flow behaviour of cerebrospinal fluid (CSF) within the central nervous system (CNS) has been the source of much discussion within the medical community. Understanding of this area has important implications for the treatment of several pathological conditions. A central area of unresolved debate concerns the driving forces for CSF motion. The relative magnitude and role of pulsation of different regions of the CNS has come under question. Modern magnetic resonance imaging (MRI) methods allow transient measurement of CSF flow velocities. This data may be used in the construction of numerical models of CSF flow. However, the small scale of flow requires high velocity resolution, resulting in reduced spatial and temporal resolution. This places limitations on the accurate formation of boundary conditions. No authors have accurately modelled pulsation of the ventricular walls. There exists therefore a need for a 4-dimensional interpolation technique to produce a continuous temporal and spatial velocity profile of CSF flow. This thesis describes a novel 4-dimensionallocal radial basis function (RBF) software tool for the interpolation of uniformly spaced, small scale MRI data. The .application of the tool is demonstrated firstly on MRI velocity data of CSF flow within the brain. The method is then extended to the interpolation of such data to the inlet boundary cell centres of a numerical model ofCSF flow, constructed from anatomical MRI data. The model is validated through comparison with the MRI results: An algorithm for the removal of rigid body motion effects due to movement of the test subject during scanning is also described. CSF flow variation in the interpolated MRI results indicates a pulsatile flow pattern with expansion and contraction of the lateral ventricles. The results of the numerical simulation show some discrepancy with the MRI data. However, peak flow velocities in the cerebral aqueduct are comparable with the literature. Due to the complex geometrical features of the ventricle system and the limitations of the interpolated MRI data, the numerical simulation has been found to be highly sensitive to the positioning of the inlet boundaries. Rigid body motion effects are found to be negligible for larger scale CSF flow but may affect ventricular wall pulsation. l

NMDA receptors are of particular importance in the control of synaptic strength and integration of synaptic activity. Dopamine receptor modulation of NMDA receptors in the striatum may influence the efficacy of synaptic transmission in the cortico-striatal pathway (Calabresi et al., 2000c Centonze et al., 2003) and if so, this modulation will be lost in Parkinson's disease. This change may be an important factor in the changes in the basal ganglia neural network that occur in Parkinson's Disease. In this thesis I have studied dopamine D1 and D2 receptor modulation of NMDA receptors in medium spiny neurons of 7-21 day old rat striatum. The dopamine D1 receptor agonist, SKF-82958, significantly decreased rat striatal NMDA receptor currents in patch-clamp whole-cell recordings from 7 day old rats. This inhibition was not abolished by application of a G protein inhibitor (GDP-p-S) or irreversible activator (GTP-y-S) suggesting a G protein-independent mechanism. In addition, intracellular application of protein tyrosine kinase inhibitors (lavendustin A or PP2) abolished D1 inhibition of NMDA currents. Functional NR2A receptors were absent in 7 day old rat striatum according to my experiments. Single-channel recordings showed that direct D1 receptor inhibition of NMDA receptors can not be observed in isolated membrane patches, which may indicate that D1 inhibition in whole-cell recordings is mediated by a change in NMDA receptor trafficking. Consistent with this hypothesis, intracellular application of a dynamin inhibitory peptide (QVPSRPNRAP) abolished D1 inhibition of NMDA receptor currents. I therefore conclude that a tyrosine kinase-dependent alteration of NMDA receptor trafficking underlies D1 dopamine receptor-mediated down-regulation of NMDA receptor currents in the striatum. The D2 class dopamine receptor agonist, quinpirole, significantly inhibited the NMDAR responses at 1 uM, but at a lower concentration (40 nM) there was no significant effect in 7 day old rat striatum. Replacement of GTP with GDP-P-S in the pipette solution abolished the inhibition induced by 1 uM quinpirole suggesting a G protein-dependent mechanism underlies the D2 family dopamine receptor modulation of NMDA receptors in the striatum.

The serotonergic system along with other brain neurotransmitter systems has been implicated in the modulation of cognitive function. Dysregulation or pathology in neurotransmitter systems is thought to underlie the cognitive impairments associated with normal ageing, a number of disease states and chronic drug abuse. Research into the influence of serotonergic systems on cognition has focussed on the modulation of other neurotransmitter systems by serotonergic input and the importance of serotonergic receptor subtypes for learning and memory. There is evidence supporting an action of serotonin to inhibit attentional processes, perhaps primarily through inhibition of dopaminergic function, but also via other neurotransmitter systems critical to attentional function such as the noradrenergic and cholinergic systems. Studies indicate that the serotonin selective reuptake inhibitors may impair aspects of attention, whilst acute tryptophan depletion to reduce serotonin synthesis and release, may enhance aspects of attention. These data have resulted in several researchers proposing general theories of serotonergic inhibition, particularly in respect to attention/arousal. However, differential effects may be seen from studies of the various serotonergic receptor subtypes, which have so far been targeted, indicating a general theory may not be sufficient to explain the data. The evidence presented in this thesis demonstrates that some of the paradigms used thus far to support general theories of serotonergic inhibition of attention/arousal may be flawed. Specifically, monoamine depletion studies may not be able to separate serotonergic and dopaminergic influences on cognition, whilst studies of selective serotonin reuptake inhibitors and chronic ecstasy use have not controlled well for influences of sleep on cognition. Furthermore, evidence from studies of the serotonin receptor subtypes may indicate effects specific to neuropsychological processes underlying measures of attention/arousal or differential effects on aspects of cognition, which may contradict a general theory of inhibition. In conclusion, general theories of inhibition are still sufficient to account for the majority of data. However, in further academic and clinical research, thorough investigation of cognition will be critical to the development of more detailed theory and the development of effective drug treatments for cognitive disorders. Furthermore, the consideration of confounding factors in research such as the influence of sleep on cognition and the competition between monoamines for transport, is critical to the understanding and interpretation of the scientific literature to date.

The broad intention of this thesis was to understand which factors regulate the release of serotonin and noradrenaline throughout the brain. Multiple factors, such as ion channels, transporters and neurotransmitter receptors shape the release of serotonin and noradrenaline within strict spatial and temporal windows. I was interested in how fast inhibition mediated by GABA_ARs may influence LC and DRN neuronal excitability. Therefore, within this thesis I localised distinct GABA_AR subunits to the cellular and sub-cellular compartments of neurochemically diverse cell types which comprise the networks of two major monoaminergic brain centres, the noradrenergic Locus Coeruleus (LC) and the serotonergic Dorsal Raphe Nucleus (DRN). The GABA_AR alpha1 subunit was predominantly localised to the non-principal, putative interneurons of the LC and DRN, whereas the GABA_AR alpha2 and alpha3 subunits were mainly localised to the principal monoaminergic cells. This apparent segregation suggests that the precise targeting of certain GABA_AR subunits to different cellular and sub-cellular compartments is important for shaping LC and DRN neuronal excitability, and thus the release of noradrenaline and serotonin. As these monoaminergic systems are engaged by stressor exposure (Swinny et al., 2010, Kirby et al., 2000, Kirby et al., 2007), and as they have been shown to have an important role in shaping mood (Stockmeier et al., 1998, Baumann et al., 2002), I was also interested to understand whether stressors engaged the GABAergic system to influence the release of monoamines. Moreover, I have demonstrated that a mild repeated stressor influences GABA_AR expression at the transcriptomic level, in a brain region and subunit specific manner and thus provide evidence for an important role of the GABA_AR alpha3 subunit in the processing of stressor related information via the DRN. The finding that the stress neuropeptide CRH, contacts putative inhibitory synapses of serotonergic and non-serotonergic neurons of the DRN provides further evidence for the potential role of GABAergic neurotransmission in shaping DRN neuronal excitability in response to stressors. Finally, through behavioural phenotyping I have been able to demonstrate that a stressor induced increase in GABA_AR alpha3 subunit expression in the DRN, parallels adaptive-like behavioural changes in response to a novel environment.

N-Methyl-D-aspartate (NMDA) receptors are critically involved in synaptic transmission, neural development and various forms of neuronal plasticity including long-term potentiation (LTP) and long-term depression (LTD). They are also involved in the production of neuronal damage following excessive activation by glutamate released as a result of hypoxia or ischaemia. Each heteromeric receptor includes one or two NRl subunits, at least two of the four NR2A-D subunits and less usually the NR3AJB subunits. This study demonstrates that the putative NR2B subunit-containing NMDA receptor antagonist Ro 25-6981 potentiates the effects ofNMDA on rat hippocampal slices. The NR2A subunit antagonist PEAQX blocks the effects of NMDA alone and the potentiated response following Ro 25-6981 application. Furthermore, Ro 25-6981 was not neuroprotective as reported previously but unexpectedly precipitated excitotoxicity. The potentiating effect of Ro 25-6981 required around 20 minutes to become apparent, took a further 30 minutes to reach its maximum effect and was irreversible. It was not prevented by staurosporine (a broad-spectrum protein kinase inhibitor), okadaic acid (a potent inhibitor of the serine/threonine protein phosphatases types 1 and 2A) or anisomycin (a protein synthesis inhibitor). However, the potentiation was prevented by cyclosporin A (an inhibitor of Ca2+/calmodulin-dependent phosphatase 2B [calcineurin]). The results indicate that in an intact neuronal network, NR2B subunits tonically gate NR2A subunit-containing receptor function by a negative coupling mechanism involving ca1cineurin activation. NMDA receptor-dependent LTP induced by high frequency stimulation was prevented by PEAQX, an NR2A antagonist. Ro 25-6981 was unable to prevent L TP induction but was associated with a marginal reduction in the magnitude of LTP induced. There is evidence for the binding of homoquinolinic acid to an NMDAinsensitive novel binding site in the brain. This study investigated the pharmacology of homoquinolinate on the evoked field excitatory synaptic potential (fEPSP) recorded from the CAl area of rat hippocampal slices. Two NMDA receptor agonists, quinolinic acid 150/lM and homoquinolinic acid 2.5/lM, caused an approximately 50% inhibition of fEPSP slope. Paired-pulse studies suggested there might be a presynaptic component to this action that is independent of presynaptic adenosine Al receptor activation. The broad-spectrum EAA antagonist kynurenic acid and the NMDA receptor blockers 2-amino-5-phosphonopentanoic acid and dizocilpine could prevent the inhibition of fEPSP slope. None of these antagonists revealed any other NMDA-insensitive activity of homoquinolinic acid. The use of 2-carboxy-3-carboxymethylquinoline (CCMQ) to displace the reported NMDA-insensitive binding had no effect on either baseline fEPSP slope or the depression caused by homoquinolinic acid. It was also apparent that responses to homoquinolinic acid were blocked completely by the NR2A subunit-selective antagonist PEAQX, but not by the NR2B subunit-selective blocker Ro 25-6981. It was concluded that the novel binding site for homoquinolinic acid does not affect synaptic potentials in the hippocampus and that homoquinolinic acid appears to be a selective agonist at NMDA receptors that include the NR2A subunit. Although the NR2B agonist site may be maximally activated under normal conditions and therefore it is not possible to observe any additional effects upon fEPSP slope. This study next investigated the negative coupling between NR2B and NR2A subunit-containing receptors, combining the NR2A1B subunit selective agonist HQA with the NR2B and NR2A selective antagonists Ro 25-6981 and PEAQX. The negative coupling observed previously with applications of NMDA was also seen using HQA and QA. The potentiation of responses to HQA by Ro 25-6981 application was also associated with an enhancement of paired-pulse interactions. The subsequent application of PEAQX was able to block both the depression of fEPSP slope and the associated enhancement of paired-pulse interactions. The presence of a presynaptic element during applications of HQA alone and potentiated responses alike and the blockade of these effects by PEAQX suggests the NR2A subunit-containing NMDA receptor is responsible for the presynaptic effects acting either directly at presynaptic sites or indirectly at postsynaptic sites leading to the raising of a retrograde signal. The NR2B subunit in both its activated and antagonised state was associated with enhancements in paired-pulse interactions which suggest that it is not able to modulate directly the presynaptic element. However, whilst paired-pulse interactions are generally accepted to he presynaptic phenomena, it does not follow that postsynaptic effects cannot influence the appearance of changes in these interactions in field recordings. The absence of any observable difference between HQA, QA and NMDA results suggests that the NR2D subunit is not obviously involved in these processes.

The dopamine signalling pathway has been implicated in the pathophysiology of neuropsychiatric conditions including bipolar disorder and schizophrenia. A detailed analysis of this pathway is essential for understanding these conditions. Previous work (Zhan et al., 2008) has identified eleven novel human dopamine receptor interacting proteins (DRIPs), but their role in cell signalling remains unclear. In this project we have employed a biomedical model, Dictyostelium discoideum, to help elucidate the cellular signalling of two DRIPs, the Zizimin GEF (DRIP2) and MARK (DRIP9) proteins. Bioinformatics analysis of these proteins shows conservation of the domain structure in the human and Dictyostelium gene products. To investigate the function of these proteins during development, two Dictyostelium homologues within each family (ZizA and ZizB; MrkA and MrkC) were ablated and changes in developmental for resulting null mutants were analysed. Development was unaltered following ablation of zizA, mrkA and mrkC, however, ablation of zizB gave rise to a clear change in developmental morphology. To further understand the developmental defect of zizB, directional cell movement (chemotaxis) was analysed in the zizA and zizB null mutants. Ablation of zizA caused no gross phenotypic change in chemotaxis, whereas zizB ablation gave rise to a reduction in cell speed, directionality and aspect (roundness). Furthermore, expression studies showed zizA and zizB were constantly expressed throughout development. Overexpression of each gene (labelled with the fluorescent tag, GFP) demonstrated a cytosolic localisation the gene products, with the ZizB-GFP fusion protein additionally exhibiting enrichment of the cortex, causing a large increase in filopodia formation and a partial inhibition of cytokinesis. Analysis of protein binding partners for ZizB indicates specific interaction with Rac1 A and a range of actin-interacting proteins. In conclusion this project provides the first insight into the molecular and cellular functions of Zizimin proteins, potential dopamine receptor interacting protein.

Bibliography: leaves 180-199. / xvii, 199, [68] leaves, [1] leaf of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Primary aim was to determine whether levels of the endogenous peptide substance P (SP) would parallel and reflect the reported increased levels of the trophic agent nerve growth factor which is associated with the development of sympathetic hyperinnervation (and ultimately hypertension) in the genetic animal model for hypertension, the spontaneously hypertensive rat. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1995

Dopamine is an important neurotransmitter that is involved in several human functions such as reward, cognition, emotions and movement. Abnormalities of the neurotransmitter itself, or the dopamine receptors through which it exerts its actions, contribute to a wide range of psychiatric and neurological disorders such as Parkinson’s disease and schizophrenia. Thus far, despite the great interest and extensive research, the exact role of dopamine and the causalities of dopamine related disorders are not fully understood. Here we have developed multimodal imaging methods, to investigate the release of dopamine and the distribution of the dopamine D2-like receptor family in-vivo in healthy humans. We use the [¹¹C]PHNO PET ligand, which enables exploration of dopamine-related parameters in striatal regions, and for the first time in extrastriatal regions, that are known to be associated with distinctive functions and disorders. Our methods involve robust approaches for the manual and automated delineation of these brain regions, in terms of structural and functional organisation, using information from structural and diffusion MRI images. These data have been combined with [¹¹C]PHNO PET data for quantitative dopamine imaging. Our investigation has revealed the distribution and the relative density of the D3R and D2R sites of the dopamine D2-like receptor family, in healthy humans. In addition, we have demonstrated that the release of dopamine has a functional rather than a structural specificity and that the relative densities of the D3R and D2R sites do not drive this specificity. We have also shown that the dopamine D3R receptor is primarily distributed in regions that have a central role in reward and addiction. A finding that supports theories that assigns a primarily limbic role to the D3R.