Anatomy and Neuroscience - Theses

Permanent URI for this collection

Search Results

Now showing 1 - 10 of 114
  • Item
    Thumbnail Image
    Exploring concepts of cardiomyocyte 'stiffness' in cardiac diastolic pathology
    Janssens, Johannes Vasilios ( 2023-12)
    Background Diastolic dysfunction is a common and early feature of cardiometabolic diseases. In patients with cardiometabolic disease it is a predictor of clinical outcomes. It typically progresses silently over a period of years in the worst case scenario resulting in heart failure. Despite this, there are no in vitro diagnostics or treatments that directly target diastolic dysfunction in this population. This may reflect absence of understanding about the combination of cellular and molecular characteristics that manifest diastolic dysfunction. At the intact heart level, diastolic dysfunction can be characterized as an impaired ability to relax and increased left ventricular stiffness. Consistent functional and molecular features of cardiomyocytes which may contribute to diastolic dysfunction in cardiometabolic disease settings remain unresolved. At the cardiomyocyte level, emerging evidence suggests that intrinsic stiffness of cardiomyocytes may be an important contributor to diastolic dysfunction while at the molecular level a sugar replete heart is a characteristic feature in cardiometabolic disease. The ability for excess sugar availability to predispose to non-enzymatic and irreversible modification (glycation) of the structure and function of the myofilament proteins responsible for cardiomyocyte stiffness and relaxation remains unexplored. Importantly, these cellular and molecular characteristics may form the basis of novel diagnostic or treatment strategies for early detection of diastolic dysfunction. The aim of this Thesis was to explore and define concepts and characteristics of cardiomyocyte stiffness and the molecular features thereof, especially as it relates to cardiometabolic disease. Research questions Question 1: Does intrinsic cardiomyocyte stiffness measured in vitro contribute to in vivo diastolic dysfunction in diet-induced cardiometabolic disease? (Chapter 2) Question 2: Are human purified troponin complex and troponins enriched from rodent myocardium modified by advanced glycation end products endogenously and are these modification sites in domains of functional relevance as described in the literature? (Chapter 3) Question 3: Can a liquid chromatography coupled MS/MS approach be developed to quantify the extent of amino acid site specific carboxymethyllysine modification on cardiac troponin I. (Chapter 4) Methods Cardiometabolic disease was induced in rodents via dietary (High fat/sugar; prediabetes or Type 2 diabetes) or pharmacological (streptozotocin; Type 1 diabetes) interventions. Echocardiography was used to assess in vivo heart function in cardiometabolic disease rodent models. Cardiomyocytes were isolated from high fat/sugar fed mice and respective controls by collagenase dissociation. Glass fibers were attached (MyoTak) at cardiomyocyte longitudinal ends, and paced cardiomyocytes (2, 4Hz, 2.0mM Calcium, 37C) were subjected to progressive stretch protocol. Force development, sarcomere length, and intracellular calcium transients (Fura2AM, 5uM) were simultaneously measured (Myostretcher, IonOptix). Liquid chromatography coupled MS/MS was used to map the occurrence of Advanced Glycation End product modifications (CML, carboxymethyllysine; CEL, carboxyethyllysine; MGH1, Methylglyoxal derived hydroimidazolone) across the human troponin complex. Western blot was used to quantify total CML amounts on myocardial proteins of interest in Type 2 diabetic human and Type 1 diabetic rat myocardial samples. A novel immuno enrichment, on-bead AspN digestion, and inclusion list assisted LC MS/MS acquisition approach was developed to quantify site specific abundance of CML modifications on myocardial proteins of interest. Results Answer 1: Intrinsic cardiomyocyte stiffness is a contributing characteristic of diastolic dysfunction in diet induced cardiometabolic disease. This shows that load and stretch are important factors to consider in assessing diastolic function of cardiomyocytes. (Chapter 2) Answer 2: The human troponin complex is modified by advanced glycation end-products at 18 lysine and arginine sites. Additionally, the advanced glycation end product carboxymethyllysine was detected at 3 sites in streptozotocin-induced Type 1 Diabetic rats but not control counterparts. These modifications were detected in regions of protein protein interaction, adjacent to phosphorylation sites and in key regions of conformational change. (Chapter 3) Answer 3: A new approach for quantifying carboxymethyllysine modified protein was demonstrated. The approach employed a combination CML-modified reference library paired with specific protein immuno enrichment and yielded a set of 4 criteria to use to validate peak selection for quantification. (Chapter 4) Conclusion This Thesis reveals novel pathologic features of cardiomyocyte function in cardiometabolic disease and links them with in vivo diastolic dysfunction. New molecular mechanisms relating to disease specific post translational modification which may underly cardiomyocyte diastolic dysfunction at the contractile myofilament were discovered. Finally, novel quantitative methodology was developed to advance the translational application of these myofilament modifications as potential diagnostic markers for the early detection of diastolic dysfunction in cardiometabolic disease. Specific information pertaining to Chapter 4 of the Thesis has been omitted from this publicly published abstract for the purpose of intellectual property protection.
  • Item
    Thumbnail Image
    The role of neurotrophic factors in osteoarthritis pain
    Nazemian, Vida ( 2023-10)
    Introduction: Osteoarthritis (OA) is a progressive disease of synovial joints and subchondral bone characterized by swelling, stiffness and pain. Brain-derived neurotrophic factor (BDNF) and artemin (ARTN) are neurotrophic factors that are important regulators of pain, and have recently been implicated in the pathogenesis of OA pain. This study aimed to explore roles for BDNF and ARTN in OA pain, by investigating whether the expression of BDNF and ARTN, and their receptors (TrkB and GFRa3), is altered in different tissues at different stages of OA, and whether blocking their signalling during late-stage OA can alleviate pain. Methods: The monoiodoacetate (MIA)-induced OA of the rat knee joint was used to explore roles for BDNF and ARTN signalling in OA pain. Pain behaviour was assessed using the dynamic weight-bearing apparatus to assay OA-induced changes in hindlimb weight bearing behaviour, at different stages of disease (early vs late). Histopathological alterations in the knee joint and surrounding bones were assayed using Haematoxylin and Eosin staining and scored using a modified OARSI scale. Changes in expression of BDNF/TrkB and ARTN/GFRa3 were explored using Western blot analysis of lysates from different tissues (joint, bone, and DRG), and at different timepoints of the disease (early vs late). The dynamic weight-bearing assay was used to determine if inhibiting BDNF signalling (with a peptide mimetic TrkB inhibitor) or ARTN signalling (with a sequestering antibody) could relieve pain at late-stage disease. Results: The results of this thesis highlight differential histopathological changes occurring in the early and late stages of OA, with joint involvement being prominent in early OA, and bone and cartilage involvement in late OA. BDNF expression was increased in the joint in early OA and in the bone in late OA. ARTN expression was also increased in the joint in both early and late OA and in the bone in late OA. Attempts to alleviate pain in MIA-injected animals by targeting the BDNF/TrkB and ARTN/GFRa3 signalling pathways did not yield pain relief outcomes with the therapeutic approach chosen in this study. Conclusion: Our findings suggest that altered pain behaviour in early MIA-induced OA is associated with changes in the joint not surrounding bones, while altered pain behaviour in late MIA-induced OA are attributable to the surrounding bones. Furthermore, BDNF and ARTN may contribute differentially to pain in early and late stages of MIA-induced OA through actions in joint versus bone. These findings support further investigations into the role of BDNF and ARTN signalling in OA pain and the development of novel targeted therapeutic approaches for managing OA pain.
  • Item
    Thumbnail Image
    Targeting lipid metabolism for prostate cancer therapy.
    Fidelito, Gio ( 2023-10)
    Deregulating cellular metabolism as a hallmark of cancer involves adaptation in nutrient acquisition, preferential utilisation of substrates, and transcriptional changes that alter intracellular metabolic signalling pathways. Understanding aspects of metabolism that are essential for cancer cells to sustain their uncontrolled proliferation can help to identify metabolic vulnerabilities, which can be therapeutically harnessed to limit cancer growth. Although prior studies in cells and mice have demonstrated the importance of oxidative metabolism and lipogenesis in prostate cancer growth and progression, however, the metabolic landscape of human prostate cancer remains unclear. Accordingly, this thesis aimed to elucidate the metabolic landscape of human prostate cancer and to identify targetable metabolic dependency for the development of novel therapeutic strategies for patients with prostate cancer. To define the metabolic landscape of human prostate cancer, we assessed substrate metabolism using radiolabelled (14C) and stable (13C) isotope tracing in precision-cut slices of patient-derived xenografts (PDXs). Glucose, glutamine, and fatty acid oxidation was variably upregulated in malignant compared to benign PDXs. De novo lipogenesis (DNL) and storage of free fatty acids into phospholipids and triacylglycerols were increased in malignant PDXs. There was no difference in substrate utilisation between localised and metastatic PDXs and hierarchical clustering revealed marked metabolic heterogeneity across all PDXs. Mechanistically, glucose utilisation was mediated by acetyl-CoA production rather than carboxylation of pyruvate, while glutamine entered the TCA cycle through transaminase reactions before being utilised via oxidative or reductive pathways. Blocking fatty acid uptake or oxidation with pharmacologic inhibitors was sufficient to reduce cell viability in PDX-derived organoids, whereas blockade of DNL, or glucose or glutamine oxidation induced variable and limited therapeutic efficacy. These findings demonstrate that human prostate cancer, irrespective of disease stage, can effectively utilise all metabolic substrates, albeit with marked heterogeneity across tumours. We also confirm that fatty acid uptake and oxidation are targetable metabolic dependencies in human prostate cancer. Considering the requirement of fatty acids in most tissues, targeting these processes may not be achievable. Therefore, we posited that identification of essential proteins of lipid metabolism, which are required for prostate cancer survival, would guide the development of novel strategies for treating prostate cancer. In Chapter 3, we developed a bespoke human lipid metabolism knockout library, performed CRISPR-Cas9-based functional genomics screening, and identified 63 commonly shared essential genes between LNCaP, C4-2B, and MR-49F. Having identified multiple genes within the mevalonate-dolichol-N-glycosylation biosynthesis to be essential, we subsequently validated the requirement of this pathway in Chapter 4. Deletion of genes encoding enzymes within this pathway resulted in reduced cell proliferation and anchorage-independent growth. This was partly mediated by G1 cell cycle arrest, induction of cell death and senescence. Further in vivo validation of genes within the dolichol-N-glycosylation biosynthesis (NUS1, DOLK, DPAGT1) in xenograft model demonstrated the requirement of NUS1 for tumour growth. Collectively, the work in this thesis highlights the heterogeneity and complexity of metabolic profiles in human prostate cancer. Furthermore, we have identified putative essential genes of lipid metabolism which may provide novel strategies for targeting lipid metabolism for patients with prostate cancer.
  • Item
    No Preview Available
    Integrated Control of Gastric Function - Contributing to the Development of The Virtual Stomach
    Di Natale, Madeleine Rose ( 2023-12)
    The stomach is the first reservoir of the gastrointestinal tract, it has been referred to as the portal to the digestive system as it is the centre which assesses and processes content before it is passed to the small intestine where the great majority of nutrient absorption occurs. Functional gastric disorders which chronically impact the motility patterns cause symptoms such as severe abdominal pain, early satiety, nausea and vomiting which can impact one’s quality of life. Current treatments for these conditions are largely ineffective and given the chronic nature of these disorders many pharmacological treatments become less effective over time. Approximately 10% of the world’s population are affected by functional gastric disorders. These disorders can be caused by changes in the extrinsic or intrinsic neuronal circuitry. Therefore, neuromodulation therapies have been seen as a promising alternative therapy. However, there are still gaps in our knowledge of the stomach anatomy and enteric integrated control which is required to effectively treat these conditions. This thesis aims to detail the anatomical structures of the stomach and map the intrinsic neuronal inputs of the stomach muscle which will fill these knowledge gaps and further our understanding of the integrated control of gastric function at the autonomic neuromuscular junction. Anatomical investigations indicate that the organisation and directions of the muscle layers in the rat are quite similar to the arrangements seen in the human stomach; however, there are some intricate features that are different to standardised textbooks. This includes the hair-pin turn of the longitudinal muscle bundles on the proximal stomach ventral and dorsal surfaces as well as the identification of the esophago-pyloric ligaments which had not been correctly identified in the literature prior to my publication (Chapter 2). The alignment between anatomical structure and function across species has also been discussed and a standardised nomenclature has been proposed in Chapter 6. The morphologies, distribution patterns and targets of the neurons were investigated and demonstrate different ganglia patterns from proximal to distal stomach and differences at the lesser and greater curvatures. These innervation differences turned out to correspond to functional innervation patterns (Chapter 3). We have demonstrated that there are multiple neural inputs to any single smooth muscle cell as increasing the stimulus voltage recruits additional inputs, recruiting axons with different thresholds which increases the junction potential amplitude that is recorded. The influence of inhibitory neurons declined with increased distance from the stimulation site. Differences in neuromuscular transmission across anatomical regions of the stomach and the influence of interstitial cells within the antrum were investigated in Chapter 5. The inhibitory neurons produced a hyperpolarising event across all three anatomical regions of the stomach. The excitatory neurons produced depolarising events in the fundus, intermittent and smaller depolarisation in the corpus, whilst depolarisation events were not recorded in the antrum. The data generated in this thesis are part of a larger collaborative initiative and will contribute to the development of a computer simulation model of the stomach which aims to realistically replicate the mechanisms and motility patterns of the stomach: This initiative is entitled The Virtual Stomach.
  • Item
    Thumbnail Image
    Using human stem cell-derived cortical neurospheres as a model to study the impact of diabetic conditions on neurodevelopment
    Mor, Michal ( 2023-06)
    In recent years, growing evidence has linked diabetic-induced dysglycemia and abnormal insulin with disordered human neural ontogeny resulting in functional deficits. Children who develop type 1 diabetes are observed to have developmental abnormalities in cognition, mental health, brain morphology and brain biochemistry. Exposure to dysglycaemic conditions during pregnancy may also have ongoing impacts on the cognition of the developing infant, including lower IQ and increased incidence of ASD and ADHD. The aim of this study was to better understand how gestational diabetes, maternal diabetes, and type 1 diabetes can affect early brain development. To achieve this, cortical neurospheres (NSPs) derived from human embryonic stem cells (hESC) were used as a platform to simulate the in-utero impact of changing glucose and insulin levels on the developmental pathways of neural stem cells during human cortex development. First, a new neural media was established to support hPSC neural induction and NSP maintenance, which more closely resembled the human central nervous system's physiological glucose and insulin concentrations. These media served as a more appropriate platform to model in-utero brain development and investigate neural differentiation signalling pathways impacted by abnormal glucose and insulin levels. Following this, in Chapter 4, I examined the effects of abnormally high glucose levels on neural differentiation in hPSC-derived neurospheres. The aim of Chapter 5 was to explore the impact of low insulin and different combinations of abnormal glucose and insulin conditions on neural differentiation in hPSC- derived neurospheres. This was to model the impact of hyperglycaemic-like and diabetic-like conditions on pathways and mechanisms involved in neuronal differentiation during early corticogenesis. Gene expression analyses, mass spectrometry proteomics, and IPA software analyses were used to investigate and identify candidate proteins and canonical pathways dysregulated in the developing cortical neurons due to altered glucose and/or insulin conditions. New findings highlighted changes in different pathways linked to mRNA translation, cell adhesion, stress responses, protein processing, metabolism and mitochondria function. However, the most significant changes were observed in proteins and pathways associated with cytoskeleton dynamics, including axonal guidance signalling, gap junction signalling, EIF2 signalling and protein ubiquitination, all of which are critical for normal brain development. These findings are significant for revealing initial key events underlying cognitive impairments associated with dysglycaemic and abnormal insulin conditions during early brain development.
  • Item
    Thumbnail Image
    Investigating the role of neuronal TrkB in remyelination
    Yoo, Sang Won ( 2023-02)
    Demyelinating diseases, such as multiple sclerosis (MS), involve damage to the fatty, proteinaceous myelin sheaths surrounding nerve fibres and subsequent development of neurological symptoms related to the damaged area. Repair of the damaged myelin can completely or partially reverse the neurological deficits, however, over time with repetitive demyelinating events and incomplete remyelination this ultimately results in the degeneration of neurons and development of permanent disabilities. Currently, there is a lack of therapies effective in promoting myelin repair. A complete understanding of the factors that promote remyelination is critical for developing therapeutic strategies for myelin repair to prevent permanent neurological disability. Many studies have focused on the potential of oligodendrocytes (the myelin-generating cells in the central nervous system) to enhance the generation of myelin. However, neurons certainly have an intimate relationship with oligodendrocytes and are well-positioned to influence the process of myelination. Previously, neuronal expression of the tyrosine kinase receptor TrkB has been shown to promote myelination in early development and during ageing. However, whether neuronally expressed TrkB receptors also influences the extent of oligodendroglial and myelin damage post a demyelinating injury, and the subsequent process of remyelination has not been elucidated. In this thesis, I generated an inducible, neuron-specific TrkB receptor knockout mouse line to investigate the role of neuronal TrkB receptors in the extent of both demyelination and remyelination following the induction of a demyelinating insult with cuprizone administration. I found that following deletion of TrkB, these mice exhibit a normally myelinated nervous system, but do however develop a motor phenotype consistent with a decrease in precise motor control. Following administration with cuprizone, I revealed that neuronal expression of TrkB is important for the differentiation of OPCs (oligodendrocyte progenitor cells) that are generated in response to the demyelinating insult. Finally, although reduced expression of neuronal TrkB did not influence the overall extent of remyelination, I found that axons with small diameter required expression of neuronal TrkB to be efficiently remyelinated. This work is the first to identify that neuronally expressed TrkB receptors have a role in OPC differentiation following demyelination. This thesis suggests there is therapeutic potential in targeting neuronal TrkB signalling to promote myelin repair, albeit this may only be specific to smaller diameter neurons.
  • Item
    Thumbnail Image
    The physiological relevance of ghrelin and dopamine D2 receptor (D2R) interactions
    Ringuet, Mitchell Ty ( 2023-05)
    Disorders of colorectal function, especially constipation, are poorly responsive to drug therapy and are a substantial medical problem. It is estimated around 15-25% of Australians over the age of 65 suffer from constipation. This presents a burden for patients and society that must be addressed. Centrally penetrant ghrelin and monoamines (dopamine, serotonin) have pro colokinetic effects, enhancing colonic motility. Agonists likely act on the same neurons in the lumbosacral defecation centre, where their cognate receptors exist, although this has not been investigated. Intriguingly, the ghrelin receptor (GHSR) but not ghrelin can be identified in this region, suggesting either an orphan ligand or an apo-ghrelin receptor function. I hypothesised that dopamine, ghrelin, and serotonin act at parasympathetic preganglionic neurons (PGNs) in the lumbosacral defecation centre, where I predict GHSR, 5-hydroxytryptamine receptors (5-HTR) and dopamine D2 receptors (D2R) exist. Furthermore, I predict that GHSR and D2R functionally interact in these neurons, although this has not been examined. Using RNAscope I identified transcript for Ghsr and Drd2 in the same PGNs (identified by Chat transcript), in the lumbosacral defecation centre in rodents (both rats and mice). I also found retrogradely labelled PGNs in neonatal rats were excited by dopamine and the GHSR agonist (capromorelin) in the same neuron. In adult Drd2-tdTomato (D2R) reporter mice the postsynaptic excitatory effect of dopamine was masked by activation of inhibitory interneurons, an effect that was reversed using TTX. In addition to this, I showed using calcium imaging that dopamine increased calcium in D2R neurons in the lumbosacral defecation centre. The ability for dopamine to excite these neurons is unusual, as D2R coupling in midbrain neurons typically liberates Gbetagamma (Galphai/o), activating GIRK channels, leading to neuronal hyperpolarisation. Using whole cell electrophysiology and pharmacology, I uncovered that excitation of D2R by dopamine was dependent on GHSR, as antagonists and inverse agonists (YIL781/JMV2959) of GHSR reduced the excitatory effect of dopamine. Furthermore, I found that these GPCRs act synergistically through the downstream mediator PLCbeta, increasing [Ca2+]i and causing neuronal depolarisation, likely via a calcium sensitive non-specific cation channel. Work in vitro has complemented this work showing that PLCbeta activation is dependent on both Galphai/o and Galphaq signalling, as the inhibition of either of these G proteins or PLCbeta in stably transfected cells containing GHSR and D2R prevents [Ca2+]i release. Additionally, high resolution microscopy revealed that these receptors do not physically interact “heterodimerise” as many groups have hypothesised. Moving forward, these results provide the basis for promising therapeutic approaches to treat not only constipation but a wide range of other diseases. Additionally, it opens up the field of GPCR biology to start investigating other tissues where this altered receptor transducer signalling might be having a physiological effect.
  • Item
    Thumbnail Image
    Characterisation of a novel liver progenitor cell marker in biliary atresia
    Leal, Marcelo Cerf ( 2022-06)
    The characterisation of liver progenitor cells (LPC) has proven to be difficult, likely due to plasticity in the regenerative/reparative processes of the liver based on the type and extent of injury. The recent development of an antibody to the GCTM-5 epitope has identified a putative liver progenitor cell population which was not previously described in the paediatric population. GCTM-5 was previously identified to strongly mark cells in the foetal ductal plate, early in the developmental pathway of both hepatocytes and cholangiocytes. This study describes the distribution of this antibody in patients with biliary atresia at time of paediatric liver transplant, and compares it to other known markers for cells with progenitor-like properties. We conducted a chart review of all patients with biliary atresia undergoing a liver transplant between 1995-2015 at the Royal Children’s Hospital, Melbourne. Multiple samples that had been stored from the explanted liver of patients with biliary atresia were stained for ENPRO-1, a second-generation antibody with greater affinity for the same epitope stained by GCTM-5. We found that the ENPRO-1 antibody most strongly marked ductular reaction cells in this human population, which is a histological description known to contain the LPC niche. It also identified undifferentiated EpCAM positive cells, most SOX9-positive cells, all NCAM-positive cells and some Lgr5-positive cells. As such, ENPRO-1 was found to mark a novel subset of liver cells of previously identified cells with progenitor properties. This is critical to validate future studies for a serum biomarker for activation of this LPC niche, as the epitope marked by the ENPRO-1 antibody is known to be secreted into blood. Unexpectedly, we found CD133/PROM1 to strongly mark almost all parenchymal hepatocytes in regenerative nodules, which has not previously been described, and may be unique to patients with end-stage liver failure due to biliary atresia.
  • Item
    Thumbnail Image
    Commitment to Serotonergic Signalling: Evolution and Distribution of the Serotonin Receptors
    Fernando, Sabrina ( 2022)
    Serotonin is a subclass of neurotransmitters and a relatively simple metabolite. The serotonin system underpins a multitude of biological functions by coupling with diverse serotonergic signalling. The effectors of serotonergic signalling are the serotonin receptors: a family of GPCRs (G protein-coupled receptors). These receptors are associated with diverse biological functions and are the primary sites of action for many psychotherapeutic drugs. We describe what commitment to serotonergic signalling entails by quantifying the diversity of this family in terms of receptor sequences, and expression patterns in the brain and systemic tissues. Sampling receptor sequences from the HTR (serotonin receptors) family, we constructed an updated phylogeny. We interpolated Bayesian priors to include key evolutionary events and to date the evolution of the family. Using RNA sequencing data, we provide the first systematic evaluation of the HTR distribution in the human body and organised the family based on their shared expression patterns. The phylogeny recapitulated early radiation of HTRs predating vertebrate evolution and demonstrated the three present clades of Galphas-coupled HTRs lack a singular ancestral node. The RNA-sequence analysis identified the systemic as well as brain-specific receptors, and reproducibly detected ten family members in the brain, which could be sub-classified by their co-expression patterns in cortical and subcortical regions. Together, the phylogenetic tree and the transcriptome map underscore the diversity of the HTR family, with multiple members evolved to activate all types of Galpha pathways specifically in the brain as well as systemically. Thus, through applying ‘omics data, the thesis outputs present the first systematic description of the family. The findings of this thesis reinforce the fact that multiple serotonin receptors are not evolutionary redundancies, but rather each receptor corresponds to specialised tissue distribution. An accurate understanding of the commonalities and contrasts among the subtypes would aid the development of subtype-specific drug targets.
  • Item
    Thumbnail Image
    Plasticity in the Enteric Nervous System
    Leembruggen, Anita Joy Louise ( 2022)
    The gut is constantly exposed to fluctuating environments and stimuli. To maintain optimal digestion, absorption of nutrients and waste disposal, the gut must adapt to these changing conditions. Within the gut wall is a network of neurons and glia known as the enteric nervous system (ENS), which regulates the major functions of the gut, including secretion, absorption, and motility. However, the ENS circuitry that underlies plasticity in the gut is unclear. This thesis aimed to explore different aspects of plasticity in the myenteric plexus using the mouse as an animal model, to increase our understanding of how changes in gut motility is regulated. In Chapter 3, the role of Group I metabotropic glutamate receptors (mGluRs) in enteric neurotransmission was investigated. Group I mGluRs are involved in synaptic plasticity in the central nervous system (CNS), but their role in the ENS has been unclear. Live Ca2+ imaging was performed on ex vivo preparations of Wnt1-cre;GCaMP6f mouse colon, where all neurons and glial cells of the ENS express the genetically encoded calcium indicator, GCaMP6f. It was observed that some myenteric neurons were activated by the Group I mGluR agonist, DHPG, exhibiting increases in intracellular Ca2+ concentration ([Ca2+]i). Responses were recorded from calbindin and nNOS-immunoreactive neurons, which mostly represent the intrinsic sensory neurons and inhibitory motor neurons of the ENS, respectively. The role of the individual Group I mGluRs (mGluR1 and mGluR5) on endogenous synaptic transmission was examined by the application of specific antagonists following electrical stimulation of interganglionic fibre tracts. In addition, an ex vivo video imaging assay of colonic motor complexes (CMCs) revealed a role for mGluR1 in the initiation of CMCs, as but no role for mGluR5 in CMC generation. These data suggest a complex role for Group I mGluRs in the ENS. Chapter 4 investigates the effect of circadian rhythm on ENS function. Colonic motility fluctuates during the circadian cycle, and gut dysfunction can occur when the circadian cycle is disrupted. This chapter investigated whether ENS function is altered during the circadian cycle in mice. Using live Ca2+ imaging, responses to the application of different neurotransmitters and neurotransmitter receptor agonists were examined. Overall, myenteric neurons had increased [Ca2+]i transients in response to the application of agonists during the dark phase (when mice are active) compared to the light phase (when mice are asleep). A separate cohort of mice were fasted for 13 hours prior to live Ca2+ experiments, and showed increased [Ca2+]i following agonist application during the light phase compared to the dark phase. This suggests that circadian neuroplasticity in the ENS can occur within a short timeframe and depends on the availability of food and feeding time. Finally, Chapter 5, explores viral transduction as a technique for understanding neuronal connectivity. Abdominal surgery was performed on mice to inject AAV9-CB7-eGFP into the distal colon. Dense labelling of GFP was observed in the distal colon myenteric plexus, near the injection site, while sparse labelling of myenteric neurons was observed in the proximal colon. GFP was observed in neurons expressing Calbindin, Calretinin, and nNOS. Some colocalisation was observed between GFP+ varicosities and Enk+ varicosities, but there was no colocalisation between GFP+ and VIP+ or CGRP+ varicosities. These data confirm that AAV9 can be used to transduce different neurochemical subtypes in the myenteric plexus, which is important for future studies investigating neuronal connectivity. Together, this thesis provides the first evidence that Group I mGluRs are involved in neural control of gastrointestinal motility in mice, and that there is short-term plasticity of myenteric neurons during the circadian cycle. Combined with preliminary observations of using AAV9 to transduce myenteric neurons, these data lay the groundwork for future studies to explore neuronal connectivity and plasticity in the ENS.