Anatomy and Neuroscience - Theses
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ItemDistinguishing features and regulatory roles of 5-HT containing enteroendocrine cells( 2022)Enteroendocrine cells (EEC) have important roles in communicating the state of the gastrointestinal (GI) tract to the rest of the body, and in signalling within the GI tract. They signal in response to nutrients and metabolites in the GI tract, potentially toxic compounds and mechanical forces. The most numerous EEC signal through 5-hydroxytryptamine (5-HT; also known as serotonin). These were also the first EEC to be identified, which was through their reactions with chrome salts, from which they gained the name enterochromaffin (EC) cells. It was at first thought that all 5-HT producing EEC were much the same. In the recent years, it has become apparent that there are functionally diverse subtypes of 5-HT containing EEC. EEC make up only approximately 1% of the intestinal epithelial cells but they are responsible for producing more than 20 peptide hormones. Of the 1% EEC population, EC cells are the most abundant cell type. I have shown that 5-HT containing EEC co-express a variety of gut hormones (Chapter 2), and their co-expression patterns are defining features that characterise them into various subpopulations based on the hormones they produce. Furthermore, EEC subpopulations express a collection of receptors that respond to different stimuli which impact their physiological effects, thus adding another layer of complexity when classifying the functional subtypes of EEC. 5-HT cells are generally depicted to be open flask-shape cells in the literature. However, one study showed an intriguing characteristic of long basal processes exhibited by some EC cells, though no study had characterised the distinct morphology of 5-HT cells in detail. Therefore, I have undertaken extensive investigations to document the morphological characteristics of 5-HT cells from the mouse stomach to rectum (Chapter 3 and Appendix A). Approximately 50% of 5-HT cells in the mouse distal colon had long basal processes, and this morphology was also observed in the gastric antrum and the rectum. These processes can reach 100 micron in length, and the abundance of this structure must serve some functional roles in the intestinal mucosa. I speculate on these in Chapters 3 and 7, and in Appendix A. An unanswered question arising from the complexity of hormone co-expression is whether co-expressed hormones could be differentially released. To address this, I examined the subcellular distribution of 5-HT and tachykinin (TK) storing secretory vesicles within the same EEC (Chapter 4). 5-HT and TK are stored in separate vesicles, and the two pools of vesicles were preferentially translocated when stimulated with glucose. In addition, duodenal 5-HT/TK cells responded differently than colonic 5-HT/TK cells under the same stimulated condition, suggesting a regional difference of EEC subpopulations. Insulin-like peptide 5 (INSL5) is co-expressed with GLP-1 and PYY in colonic L cells, and I discovered that some 5-HT cells had an intertwining relationship with L cells in the mouse large intestine (Chapter 3). In Chapter 5, I describe the development of an LC/MS assay for an INSL5 analogue that I used to investigate the role of INSL5 (Appendix B). I also discovered that RXFP4, a natural receptor for INSL5, is extensively expressed by colonic 5-HT cells and by some sensory nerve fibres in the mucosa, submucosa, and the muscle layers of the large intestine (Chapter 6). Hence, INSL5 could have an effect on both RXFP4 expressing nerve terminals and on neighbouring 5-HT cells. The regulatory role of 5-HT in the control of colorectal propulsion was demonstrated to be through an INSL5/RXFP4/5-HT/5-HT3R neuro-endocrine circuit. Collectively, my studies presented in this thesis have systematically defined the distinguishing features of 5-HT containing EEC throughout the GI tract and the regulatory role of 5-HT in colonic motility.
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ItemEnteric serotonin interneurons: connections and role in intestinal movement( 2008)5-HT powerfully affects gastrointestinal function. However, the study of these effects is complicated because 5-HT from both mucosa and a subset of enteric neurons acts on multiple receptor subtypes in enteric tissues. The role of neural 5-HT has been difficult to isolate with current techniques. This thesis aimed to elucidate the role of 5-HT neurons in motility using anatomical and functional methods. In Chapter 2, confocal microscopy was used to examine over 95% of myenteric neurons in guinea pig jejunum, categorized neurochemically, to identify neurons that received anatomically-defined input from 5-HT interneurons. The data showed that cholinergic secretomotor neurons were strongly targeted by 5-HT interneurons. In another key finding, excitatory motor neurons were surrounded by 5-HT terminals; this could provide an anatomical substrate for the descending excitation reflex. Subgroups of ascending interneurons and neurons with immunoreactivity for NOS, were also targeted by 5-HT interneurons. Thus, subtypes of these neurons might act in separate reflex pathways. Despite strong physiological evidence for 5-HT inputs to AH/Dogiel type II neurons, few contacts were identified. In Chapter 3, the confocal microscopy survey was extended to the three other interneuron classes (VIP/NOS and SOM descending interneurons; calretinin ascending interneurons) of guinea pig small intestine. A high degree of convergence between the otherwise polarized ascending and descending interneuron pathways was identified.
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ItemA study of depression in Huntington's disease( 2008)Huntington’s disease (HD) is an inherited neurodegenerative disorder that is caused by a mutation of a single gene, huntingtin. The disease is more commonly known for the characteristic choreiform movements that develop in the later, more advanced stages of the disease. However, cognitive deficits and psychiatric symptoms are frequently observed prior to the onset of the motor symptoms. Little is known about the pathological bases for the neuropsychiatric features which include increased irritability and heightened aggression. Depression affects 30-50% of HD patients and is the most commonly diagnosed psychiatric symptom. This is proportionally higher than in the general population and it is possible that inherent pathological changes in the HD brain render a HD-gene positive individual more susceptible to depression. Using a variety of behavioural tests, the R6/1 transgenic mouse model of HD was found to display altered responses reflective of depression-related behaviour, indicating that the HD mutation confers a genetic susceptibility for developing depression. The behavioural alterations were more robust in female HD mice reflecting a possible sex-dependent manifestation of the depression symptoms in the human HD population that has yet to be investigated. The onset and rate of progression of HD is strongly influenced by the environment and the development of depression is similarly impacted upon by environmental factors (e.g. stress, negative life events). The experimental paradigms of environmental enrichment and wheel-running slow the development of motor and cognitive symptoms in R6/1 HD mice and the present study reports that both paradigms also correct the depression-related behavioural phenotype. This study also found that HD mice had muted responses to two common classes of antidepressant drugs, highlighting the need for a detailed examination of the efficacy of drug treatments in HD patients. Depression susceptibility is linked to genetic variance in the human population and studies of gene candidates in mutant mice report the detection of behavioural phenotypes similar to the present study. The depression-related behavioural phenotype of the R6/1 HD model was found to be associated with early down-regulations in mRNA levels of the ii serotonin (5-HT) 1A and 5-HT 1B receptors in the cortex and the hippocampus. Additionally, female HD mice had reduced cortical 5-HT transporter gene expression. Collectively, these findings indicate that a disruption of serotonergic signaling in the HD brain contributes to the development of depression in HD. Brain-derived neurotrophic factor (BDNF) gene expression is down-regulated in the HD brain, however the expression pattern of exon-specific splice variants was previously unknown. This study reports that BDNF mRNA levels are reduced in the hippocampus by an early age but also reports that individual exon-specific transcripts are differentially down-regulated in males and females, although the functional relevance of this remains to be investigated. Overall, this study has demonstrated that the R6/1 transgenic mouse model of HD is ideal for further investigating the occurrence of depression in pre-motor symptomatic HD. It has also identified alterations in gene expression of key components of neuronal signaling which might be linked to the molecular basis of depression.