published_or_final_version / abstract / toc / Molecular Biology / Master / Master of Philosophy

There is evidence that the excitotoxin quinolinic acid (QUIN) synthesized through the kynurenine pathway (KP) by activated microglia may play a role in the pathogenesis of several major neuroinflammatory diseases and more particularly in Alzheimer??s disease (AD). The hypothesis of this project is QUIN affects the function and morphology of astrocytes. In this study I used human foetal astrocytes stimulated with AD associated cytokines including IFN-gamma, TNF-alpha, TGF-alpha and different concentrations of QUIN ranging from low physiological to high excitotoxic concentrations. I found that QUIN induces IL-1beta expression in human astrocytes and subsequently, contribute to the inflammatory cascade that is present in AD pathology. Glial fibrillary acid protein (GFAP) and vimentin protein expression were complementary in expression to each other after 24 hr stimulation with different QUIN doses. However, there were marked increases in GFAP levels and reduction in vimentin levels compared to controls with QUIN treatment indicating that QUIN can trigger astrogliosis in human astrocytes. Glutamine synthetase (GS) activity was used as a functional metabolic test for astrocytes and I found a dose-dependent inhibition of GS activity by QUIN. This inhibition was inversely correlated with iNOS expression whereby reduced GS activity is accompanied with an increase expression of iNOS in human astrocytes. These results suggest that reduction in GS activity can lead to accumulation of extracellular glutamate then leading to exacerbated excitotoxicity via NMDA receptor over-activation and ultimately neuronal death. PCR array results showed that at least four different pathways were activated with pathological concentration of QUIN including p38 MAPK that is associated with pro-inflammatory cytokine production, ERK/MAPK growth and differentiation that can modulate structural proteins, mitochondrial-induced apoptotic cascade and cell cycle control pathway. QUIN-induced astrogliosis and excitotoxicity could lead to glial scar formation and prevention of axonal growth thus exacerbation of neurodegeneration via synaptosomal NMDA receptor over-activation. All together, this study showed that, in the context of AD, QUIN is an important factor for astroglial activation, dysregulation and death, which can be mediated by the previously mentioned pathways.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 209-226) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 209-226) Also available online.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 98-112).

Thesis (Ph. D.)--University of Virginia, 2005. / CD-ROM has .tiff and .mov files. Includes bibliographical references (leaves 129-132). Also available online through Digital Dissertations.

The spatial location of cells in neural tissue can be easily extracted from many imaging modalities, but the information contained in spatial relationships between cells is seldom utilized. This is because of a lack of recognition of the importance of spatial relationships to some aspects of brain function, and the reflection in spatial statistics of other types of information. The mathematical tools necessary to describe spatial relationships are also unknown to many neuroscientists, and biologists in general. We analyze two cases, and show that spatial relationships can be used to understand the role of a particular type of cell, the astrocyte, in Alzheimer's disease, and that the geometry of axons in the brain's white matter sheds light on the process of establishing connectivity between areas of the brain. Astrocytes provide nutrients for neuronal metabolism, and regulate the chemical environment of the brain, activities that require manipulation of spatial distributions (of neurotransmitters, for example). We first show, through the use of a correlation function, that inter-astrocyte forces determine the size of independent regulatory domains in the cortex. By examining the spatial distribution of astrocytes in a mouse model of Alzheimer's Disease, we determine that astrocytes are not actively transported to fight the disease, as was previously thought. The paths axons take through the white matter determine which parts of the brain are connected, and how quickly signals are transmitted. The rules that determine these paths (i.e. shortest distance) are currently unknown. By measurement of axon orientation distributions using three-point correlation functions and the statistics of axon turning and branching, we reveal that axons are restricted to growth in three directions, like a taxicab traversing city blocks, albeit in three-dimensions. We show how geometric restrictions at the small scale are related to large-scale trajectories. Finally we discuss the implications of this finding for experimental and theoretical connectomics.

Addiction is a chronic relapsing disorder for which there is no effective treatment. This may reflect our lack of understanding of the psychological and neural mechanisms that support the transition, in vulnerable individuals, from recreational drug use to compulsive drug seeking habits. Over the last decade clinical and preclinical studies have begun to shed light on the psychological and neural basis of the individual vulnerability to cocaine addiction, but despite the epidemic in opiates addiction in the USA and incremental opioid drug abuse and addiction in the UK, heroin addiction has hitherto been under-investigated. Using a novel preclinical model of compulsive heroin seeking behaviour in which some rats self-administering heroin persist in responding under a second-order schedule of reinforcement despite punishment (Chapter 3), the experiments in this thesis investigated the psychological, behavioural, neural and cellular mechanisms involved in the vulnerability to develop compulsive heroin seeking. Chapter 4 aimed to identify behavioural traits, such as anxiety, stress reactivity or decision making, that predict an increased vulnerability to develop compulsive heroin seeking. Chapter 5 aimed to characterise the neural and cellular correlates of heroin seeking habits, and compulsivity. Based on the combination of hotspot analysis, quantitative PCR, RNAscope and western-blot analyses, the data presented demonstrate that compulsive habits are associated with a differential pattern of cellular plasticity within corticostriatal networks, and are preceded by diverse cellular adaptations, especially in the striatum, in vulnerable individuals. Finally, chapter 6 further investigated the cellular specificity of the observed adaptations in experiments that revealed exposure to heroin and cocaine, triggers a downregulation of the dopamine transporter preferentially in astrocytes, and not in neurons as previously thought. The results presented in this thesis offer new insights into the neural and cellular basis of the vulnerability to develop compulsive heroin seeking, a key feature of opioid addiction.

The hippocampus (HPC) is a brain area in the medial temporal lobe involved in spatial navigation, as well as the formation of episodic memories. A subset of the principal cells of the HPC, known as place cells, are active in specific locations of an environment, called the place fields. Dorsal hippocampal area CA1 contains place fields that are known to change their firing during spatial tasks where animals learn the location of a reward, known as goal-oriented learning (GOL) – CA1 place fields shift toward rewarded locations. Previous studies suggest that this preferentially occurs at novel rewarded locations in a familiar environment, but the mechanism is unknown. The locus coeruleus (LC) is a neuromodulatory nucleus in the brainstem that projects throughout the brain and releases norepinephrine and a small amount of dopamine. Stimulating locus coeruleus-hippocampal area CA1 projections (LC-CA1) was recently shown to improve performance on spatial memory tasks. Since performance on the GOL task is correlated with the degree of overrepresentation of rewarded locations, we hypothesized that the LC-CA1 projection was involved in reward-related place field reorganization. Using in vivo two photon calcium imaging, we recorded the activity of the LC-CA1 projection during a head fixed GOL task with two phases – during the first phase, a water reward was presented in one location (RZ1), and in the second phase, it was moved to a novel location (RZ2). In the first phase of the task, the LC-CA1 axons were correlated with running, but in the second phase they showed an increase in activity preceding RZ2. To determine whether the LC-CA1 is involved in place field reorganization that normally occurs in RZ2, we optogenetically activated the projection just before RZ1, and saw a pronounced place field reorganization right before the reward. Conversely, inhibition of LC-CA1 at RZ2 attenuated place field reorganization at this site. Finally, LC-CA1 stimulation away from the reward did not lead to place field reorganization, indicating that the LC influences place field shifts in conjunction with other signals that are differentially active around rewards. A full account of the effects of neuromodulation should also include astrocytes, since they respond to neuromodulators with large calcium signals that may be able to affect the function of neurons. We also recorded HPC astrocyte calcium activity during different behavioral tasks. Astrocytes showed occasional large calcium signals, with some differences in synchronicity and activity levels between hippocampal layers and behavioral paradigms. Future studies should determine whether the LC-CA1 projection affects place fields directly by affecting neural activity, indirectly via astrocytes, or both.

Opioids are gold-standard analgesics for pain relief in chronic pain conditions. Paradoxically, chronic opioid use causes an enhanced pain sensitivity termed ‘Opioid-induced hyperalgesia’ (OIH). OIH is a clinically relevant problem associated with the use of opioids. In addition to decreasing quality of life, increased pain from OIH necessitates increasing dosages of analgesics to effectively control the pain, resulting in an increased risk of opioid epidemics, addiction, and overdose. To prevent this clinically important effect, it is necessary to understand how chronic opioid use causes hyperalgesia. Our preliminary studies revealed that synaptic plasticity in the spinal dorsal horn (SDH) is dependent on neuron type in the OIH model and occurs concurrently with hyperalgesia, suggesting central sensitization as a mechanism of OIH. We found that astrocyte ablation blocked mechanical hyperalgesia and neuron type-dependent synaptic plasticity, indicating that astrocytes are critically involved in OIH. Additionally, morphine treatment upregulated IL-1β expression in the SDH in our preliminary experiments. Inhibition of IL-1β prevented OIH and blocked the repeated morphine-induced synaptic plasticity in the SDH, suggesting IL-1β is a key player in the pathogenesis of OIH. Astrocytes and other glial cells are critical in the development and maintenance of neuroinflammatory conditions, such as OIH, through the release of proinflammatory cytokines (PICs), including IL-1β. The mechanosensitive ion channel, Piezo1, was recently found to be upregulated in astrocytes and microglia under LPS-induced inflammatory conditions, and activation of Piezo1 was found to reduce IL-1β expression in LPS-inflamed primary mouse astrocytes. The goal of this study was to investigate the function of Piezo1 as a potential treatment for neuroinflammatory diseases of the CNS in a model of LPS-induced inflammation. In this study, we created a culture cell model of LPS-induced astrocytic neuroinflammation using the C8-S type II astrocyte culture cell line. We used a multi-disciplinary approach of electrophysiology and imaging to assess changes in calcium flux induced by the selective Piezo1 agonist, Yoda1, and mechanosensitive ion channel activity in the LPS-stimulated C8-S culture astrocytes. We found that calcium flux is increased in LPS stimulation and augmented by additional Yoda1 treatment. We also found that LPS stimulation increases mechanosensitive ion currents and stiffens cell membranes using patch-clamp electrophysiology techniques. These results indicate that Piezo1 is likely upregulated in the LPS model of cultured astrocytes, thus mechanosensitive responses are increased. Results from these experiments reveal key information about the mechanical properties of Piezo1 and poise Piezo1 as a promising therapeutic for OIH and other neuroinflammatory diseases caused by astrocytic IL-1β release.