Physiology - Theses
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ItemThe effect of early life antibiotic exposure on the development of the gut microbiota and enteric nervous system( 2019)Early postnatal life is a critical stage of microbiota establishment and ENS development. While the initial postnatal stage from birth is fundamental for the development of the gut microbiota and ENS, weaning is another key developmental period where there are major changes in diet, behaviour and physiology, and notably, microbiota. Antibiotics are frequently administered to infants and young children, however, recent studies have identified prospective long-term health consequences of early life antibiotic exposure on the developing gut microbiota. Yet, how antibiotic influences short and long-term ENS development remains unclear. Vancomycin is given as a prophylactic to preterm babies and paediatric patients to treat and prevent infections. It is also one of the most commonly used antibiotics on its own or as part of a cocktail in research to induce dysbiosis in mice. The aim of my PhD was to examine how early life exposure to vancomycin during two critical developmental periods affects microbiota and ENS development and whether changes observed during early postnatal life have long-term repercussions. In chapter 3, I investigated if acute administration of vancomycin, during the early postnatal period, influenced gut microbiota and ENS development. A single regimented dose of either water or vancomycin was administered daily to Wnt1-Cre;R26R-GCaMP3 mouse pups from postnatal (P) day 0 to P10/11. These mice contain a genetically-encoded fluorescent Ca2+ indicator in all enteric neurons and glia. At P10/11, vancomycin-fed pups showed significant dysbiosis, reduced myenteric neuron density and altered nNOS and calbindin neuronal subtype proportions compared to water-fed littermates. Using Ca2+ imaging, I showed that vancomycin-fed pups had more neurons responding to electrical stimulation applied to interganglionic connectives and larger amplitudes of train-evoked [Ca2+]i transients. These changes in the ENS contributed to dysmotility of the colon of vancomycin-fed pups. In contrast to the colon, the structure of the ENS and motility patterns of the duodenum were not affected by vancomycin, ruling out drug toxicity effects. P10/11 vancomycin-fed pups also had lower numbers of serotonin (5-HT) positive cells in the colonic mucosa. Altered 5-HT metabolism in these animals were confirmed by performing mass spectrometry on 5-HT biosynthesis intermediates, showing reduced concentrations of the 5-HT metabolite, 5-HIAA and droplet digital PCR (ddPCR) revealing increased gene expression of the 5-HT transporter, SERT. Bypassing tryptophan hydroxylase, by supplementing vancomycin-fed pups with 5-HTP, restored 5-HIAA levels in the colonic mucosa and prevented some of the vancomycin-induced effects on myenteric neurons, colonic motility and gut microbiota. Therefore, vancomycin exposure during the neonatal period induced significant developmental changes to both the gut microbiota and ENS. Some of these changes could be mediated by altered mucosal serotonergic signalling. In Chapter 4, I examined if vancomycin-induced changes on the gut microbiota and ENS observed at P10 were long-lasting. Newborn mouse pups were only treated with water and vancomycin till P10, then pups were left to grow to adulthood. 6-week-old mice given neonatal vancomycin had enlarged caeca, which is an indication of dysbiosis. This suggests that the gut microbiota of vancomycin-fed mice was not fully recovered despite cessation of antibiotic treatment. Adult mice treated with neonatal vancomycin had sustained reduction in myenteric neuron density. However, alterations in the proportions of nNOS+ and calbindin+ neurons observed during the neonatal periods was now restored. In contrast to the heightened [Ca2+]i activity at P10s, adult mice given neonatal vancomycin had lower numbers of neurons responding to electrical stimulation and no change in the amplitudes of electrically-evoked [Ca2+]i transients in their myenteric neurons compared to water-fed controls. Furthermore, there were no treatment-induced changes in colonic motility. Interestingly, faecal water content, which was unaffected in vancomycin-fed pups at P10, was lower in adult mice given neonatal vancomycin compared to controls. These findings indicate that although vancomycin treatment is terminated, the gut microbiota is not fully recovered and significant re-modelling of the ENS occurs, some of which are distinct to changes observed during the neonatal period. In Chapter 5, I explored the effects of vancomycin exposure between weaning and adulthood. From the day of weaning, mice were administered vancomycin or sterile water in their drinking bottles for three weeks. At 6-weeks of age, vancomycin-treated mice had dysbiosis accompanied with enlarged caeca. Similar to vancomycin-treated neonates in Chapter 3, increased synaptic activity exhibited by enteric neurons were mainly observed by larger amplitudes of train-evoked [Ca2+]i transients and increased number of neurons responding to electrical stimulation. However, in contrast to antibiotic exposure during the neonatal period, vancomycin-treated mice displayed significantly slower colonic motility, increased faecal water content and a decrease in the proportions of ChAT+ cholinergic neurons including calbindin and neurofilament-M subtypes in the myenteric plexus of the colon. Moreover, vancomycin treatment between weaning and adulthood had no effects on the serotonergic system in the colonic mucosa. Collectively, these findings suggest that vancomycin exposure from weaning had differential effects on the gut microbiota and ENS compared to administration of the antibiotic during the neonatal period. Together, my study is the first to identify and compare effects of antibiotic exposure on the gut microbiota and ENS during two critical stages of development. While vancomycin did not deplete bacterial diversity and abundance, it caused profound shifts in microbial composition in both developmental periods. Additionally, acute vancomycin exposure in both periods, resulted in dysmotility and alterations of the neuronal circuitry. Although the effects on colonic motility for mice given neonatal antibiotic treatment did not appear to be long-lasting, changes in the ENS and disrupted faecal and caeca weights, which manifested only in adulthood, suggests that early life exposure to antibiotics can have other long-term consequences on microbiota and host gut physiology.
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ItemCharacterising cellular and molecular mechanisms of cardiac diastolic dysfunction( 2019)Background: Diastolic dysfunction is an important contributor to many cardiac pathologies including diabetic cardiomyopathy and heart failure with preserved ejection fraction. It is characterised by ventricular stiffness, inadequate filling of the ventricles and elevated ventricular pressure. In addition to extracellular influences, evidence suggests that a cardiomyocyte specific intrinsic stiffness may also be an important contributor to diastolic dysfunction, but the mechanisms are not well understood. This might be partly due to the lack of specific animal models available to study underlying mechanisms, in particular in HFpEF. The aim of this thesis was to evaluate cellular and molecular mechanisms of diastolic dysfunction in a model of type 1 diabetes and in a newly characterised model of HFpEF, the Hypertrophic Heart Rat (HHR). Research questions: Q1. Can measurement of in vitro intact cardiomyocyte stiffness be correlated with in vivo diastolic function to determine whether cellular stiffness contributes to cardiac diastolic dysfunction in pathological settings? (Chapter 2) Q2. What are the subcellular mechanisms that contribute to increased stiffness in a pathological model of diastolic dysfunction? (Chapter 3) Q3. Can the Hypertrophic Heart Rat be used as a novel rodent model of HFpEF and what is the underlying cardiomyocyte pathophysiology driving diastolic dysfunction in HFpEF? (Chapter 4) Methods: Type 1 diabetes was induced in Sprague Dawley rats using a single dosage of Streptozotocin. The Hypertrophic Heart Rat (HHR) was characterised and utilised as a model of HFpEF. Echocardiography was used to assess in vivo heart function in diabetic and HFpEF rats. Surface electrocardiogram recordings were performed to assess in vivo electrical activity in HFpEF rats. Cardiomyocytes were isolated by collagenase dissociation. Under loaded conditions glass fibers were attached (MyoTak) at the cell longitudinal surface, and paced cardiomyocytes (1Hz, 2.0mM Ca2+, 37°C) were serially stretched (011.2%, piezomotor). Force development and intracellular Ca2+ transients (Fura-2AM, 5µM) were simultaneously measured (Myostretcher, IonOptix). In the HHR, histological analysis was undertaken to evaluate collagen deposition. Intracellular Ca2+ and contractility was measured in single unloaded cardiomyocytes (4Hz, 2.0mM Ca2+, 37°C). Left ventricular tissue was homogenised and used for Western blot analysis of Ca2+ handling proteins. Results: A1. Validation of in vivo and in vitro methodologies for the measurement of cardiomyocyte and cardiac diastolic function along with confirmation that in vitro cardiomyocyte stiffness directly correlates to in vivo cardiac dysfunction. This verifies the contribution of cellular stiffness to cardiac diastolic dysfunction in the pathological setting. (Chapter 2) A2. Cardiomyocyte stiffness was shown to be an important contributor to diastolic dysfunction in the diabetic heart. The additive contribution of myofilament cooperativity reduction and slowed Ca2+ reuptake were found to be the subcellular mechanisms for the intracellular stiffness. (Chapter 3) A3. A new model of HFpEF was successfully characterised which closely mirrors clinical pathology without surgical or drug intervention. Animals display early mortality, with cardiac diastolic dysfunction, preserved ejection fraction and arrhythmias. The cardiomyocyte pathology was one of hypercontractility and Ca2+ overload, contrasting strongly with what has been reported in systolic failure leading to potential new therapeutic targets for HFpEF treatment. (Chapter 4) Conclusion: This thesis demonstrates that intact cardiomyocyte stiffness contributes directly to cardiac diastolic dysfunction, which was validated in two separate pathological models. Importantly, this is the first evidence that there is an increase in the slope of the end diastolic force length relation in intact diabetic cardiomyocytes indicating increased cellular stiffness. This was linked to changes in Ca2+ reuptake during relaxation and reduced myofilament cooperativity. In addition, a newly characterized model of HFpEF was described, along with cellular and molecular changes that are apparent in this model of diastolic dysfunction, providing new insight and potentially leading to new therapeutic targets to treat HFpEF. Taken together, this thesis advances the mechanistic understanding of the cellular and molecular mechanisms of diastolic dysfunction.
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ItemSynaptic mechanisms and function in the mouse enteric nervous system( 2018)Virtually all functions of the enteric nervous system (ENS) rely on synaptic transmission, which occurs at specialised sites referred to as synapses. Molecular mechanisms behind synaptic transmission at central synapses have been extensively characterised, studies accordingly show that pre- and post-synaptic proteins localized to these synapses regulate transmission. However, little is known about the synaptic machinery involved in regulating excitatory transmission at enteric synapses. There is growing evidence to suggest that patients with synaptic protein associated neurodevelopmental and neurodegenerative diseases, such as Parkinson’s Disease, also display abnormalities in gastrointestinal function. This suggests there is a commonality between the central nervous system (CNS) and the ENS. Excitatory transmission within the ENS is primarily mediated by acetylcholine (ACh) acting on nicotinic receptors, there are also many other putative excitatory neurotransmitters in the system whose roles remain elusive. Therefore, the aim of my PhD thesis was to elucidate molecular and pharmacological mechanisms underlying excitatory transmission in the ENS. In Chapter 2, I localized synaptic vesicle proteins synaptophysin, synaptotagmin-1 and vesicular acetylcholine transporter (vAChT) to enteric varicosities. I developed two high-throughput analysis methodologies to quantify co-expression in varicosities and their close contact with enteric neurons. Using these analysis tools, I found that synaptic vesicle proteins synaptophysin and synaptotagmin-1, described to be ubiquitous in pre-synaptic terminals, are not found in all cholinergic varicosities (vAChT+) in the myenteric plexus. I found that in the submucosal plexus, all cholinergic varicosities contained synaptophysin, but some lacked synaptotagmin-1. This highlights the sensitivity of the analysis tool developed and the disparity in synaptic protein localization at cholinergic varicosities between the two plexuses. Additionally, using 3D rendering I examined close contacts between varicosities expressing synaptophysin and vAChT on neuronal nitric oxide synthase (nNOS+) neurons. I found that nNOS+ neurons receive three distinct classes of input. This includes varicosities that either contain vAChT, synaptophysin or both. Overall, my findings demonstrate that there is molecular heterogeneity in cholinergic varicosities within the ENS, which will likely transpire into distinct modes of cholinergic transmission or ACh release at enteric synapses. Moreover, this study highlights the use of advanced image analysis tools to examine connectivity and mechanisms of transmission within the ENS. In Chapter 3, I described the expression of post-synaptic density protein PSD93 in the ENS using immunohistochemical methods. I found that most myenteric neurons, including subpopulations of cholinergic and nitrergic neurons express PSD93. The wide spread expression of PSD93 in the cytoplasm and axons of enteric neurons indicates that it is an unsuitable marker for identifying excitatory post-synaptic densities in the myenteric plexus. Instead, PSD93 is likely to be involved in other cytosolic processes in addition to any role as a post-synaptic density protein at excitatory synapses. In Chapter 4, I demonstrate importance of α-synuclein (α-Syn) in cholinergic function within the ENS. α-Syn is a synaptic vesicle protein pathologically linked to neurodegenerative diseases. I show that α-Syn is expressed in varicosities and some neuronal somata within the mouse colon, a result described previously in other species. Using the quantitative method described in Chapter 2, I found that most cholinergic varicosities (vAChT+) contained α-Syn. I also investigated the implications of α-Syn deletion for ENS function using α-Syn knock out (KO) mice. α-Syn KO mice have increased proportions of cholinergic neurons in the myenteric plexus. Additionally, cross-sections of mouse colon preparations also show that α-Syn KO mice have increased cholinergic innervation to the circular muscle. Calcium (Ca2+) imaging studies reveal that fast synaptic transmission mediated by nicotinic receptors is increased in α-Syn KO mice. However, I found that α-Syn KO mice have a reduced incidence of spontaneous circular muscle contractility, suggesting that there are changes in the circuitry underlying motor patterns. Collectively, these findings suggest that there are alterations in the enteric neural circuitry of α-Syn KO mice and that α-Syn is important for cholinergic transmission. In Chapter 5, I used Ca2+ imaging and high-resolution microscopy to elucidate the mechanisms behind glutamatergic transmission within the ENS. Thus far there is conflicting evidence to suggest the involvement of ionotropic receptors and metabotropic glutamate receptors (mGluRs) in synaptic transmission. I show that many myenteric varicosities that contain vesicular glutamate transporter 2 (vGluT2) are non-cholinergic and express synaptic vesicle proteins synaptophysin using tools I developed in Chapter 2. Using 3D rendering I showed that calbindin (calb+) neurons receive more vGluT2 varicosities than nNOS+ neurons. Exogenous application of glutamate predominantly excites calb+ neurons in the myenteric plexus. Calb+ neurons also receive slow synaptic transmission mediated by endogenous release of glutamate excited by a train of electrical stimuli. Using ionotropic and group I metabotropic glutamate receptor (mGluR) antagonists, I found that group I mGluRs are involved in mediating slow synaptic transmission. This study demonstrates a role for glutamate in mediating excitability of myenteric calb+ neurons. Overall, I have developed powerful methodologies that will provide valuable tools to contribute to understanding mechanisms underlying excitatory transmission within the ENS. The molecular heterogeneity of cholinergic varicosities identified in this thesis, provides a foundation for elucidating ACh release at enteric synapses. I have also shown that post-synaptic density markers that identify excitatory synapses in the autonomic nervous system (ANS) are unsuitable for labelling excitatory synapses in the ENS. This indicates that mechanisms underlying excitatory transmission could differ between the ANS and ENS. I have highlighted the difficulty in establishing a marker for post-synaptic densities within the ENS, which is necessary for a detailed understanding of excitatory transmission. Moreover, I have shown that α-Syn is associated with cholinergic synapses and the deletion of the synaptic vesicle protein has consequential effects on cholinergic transmission and function, thus implicating α-Syn in gastrointestinal pathophysiology. I have also identified a role for group I mGluRs in mediating excitatory slow synaptic transmission, indicating that glutamate is an excitatory neurotransmitter within the ENS. These findings provide a foundation for future analyses of synaptic function in the ENS and point to key questions for further investigation of this understudied nervous system.
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ItemThe metabolic microenvironment regulates myogenic fate decisions through altered histone acetylation( 2018)1. Skeletal muscle has an extensive capacity for regeneration, a property conferred on this tissue by a resident population of skeletal muscle stem cells (MuSCs). Any impairment to this population of stem cells can lead to increased morbidity and mortality. MuSCs normally exist in a quiescent state marked by the paired box transcription factor Pax7. In response to an activating signal, MuSCs rapidly undergo a process of activation and cell-cycle re-entry. At this early stage, MuSCs begin to express the myogenic determination factor MyoD and initiate the process of commitment to the myogenic lineage. Importantly, a sub-population of MuSCs will return to quiescence so as to prevent depletion of the stem cell pool. This decision for MuSCs to either undergo commitment or self-renewal remains ill defined. Studies in cancer, and developmental and stem cell biology has identified cellular metabolism as playing a key role in directing changes associated with stem cell self-renewal, lineage specification and the processes of proliferation and differentiation. Therefore, the aim of this study was to investigate this link between metabolism and the processes of self-renewal and myogenic commitment in MuSCs and to identify how metabolism may regulate these processes. 2. To efficiently isolate a large number of primary MuSCs I utilized Pax7creERT2xROSA26eYFP transgenic mice which allows for the fluorescent labelling of MuSCs and subsequent isolation via fluorescence activated cell sorting. To examine the link between innate cell metabolism and MuSC heterogeneity, single cell RNA sequencing (scRNAseq) was performed on either freshly isolated MuSCs or MuSCs that had been cultured ex vivo for 96 hrs. The scRNAseq results revealed that while freshly isolated MuSCs are largely homogeneous, cultured MuSCs exhibited significant heterogeneity with divergent metabolic signatures. These metabolic signatures marked cells either undergoing myogenic commitment or self-renewal. 3. To examine the role of the metabolic microenvironment in regulating MuSC lineage specification whole skeletal muscle fibres, isolated primary MuSCs or C2C12 cells were cultured in media containing different carbohydrate conditions; high glucose (25 mM glucose, HG), low glucose (5 mM glucose, LG) or galactose (10 mM galactose, GAL). Following culture, the downstream effects on metabolism, including measurements of mitochondrial DNA, mitochondrial abundance, key electron transport chain proteins and cellular bioenergetics was assessed. Myogenic specification was assessed via examination of key myogenic regulatory factors by PCR, western immunoblotting, immunofluorescence, whole transcriptome sequencing and single cell sequencing (scRNAseq). Finally, to link alterations to metabolism to changes in gene transcription, global histone acetylation was examined. Extracellular carbohydrate availability directly regulates both innate cellular metabolism and gene expression via acetyl-CoA availability and histone acetylation. Importantly, use of several pharmacological modulators of metabolism confirm a central role of carbohydrate metabolism in histone acetylation. Combining both whole transcriptome sequencing and scRNAseq, extracellular carbohydrate availability was shown to directly influence lineage fate decisions, with reduced carbohydrate availability linked to a reduction in the proportion of cells undergoing myogenic commitment. The scRNAseq dataset presented provides entirely new information of subpopulations of cells; including true MuSCs, primed MuSCs, early committed muscle progenitors (CMPs) and late CMPs and show that the extracellular metabolic environment directly influences the proportion of cells in each of these subpopulations. Finally, single fibre experiments showed that reduced carbohydrate availability was linked to increased rates of asymmetric division and self-renewal. 4. These results provide the first evidence that the extracellular metabolic microenvironment is able to directly alter MuSC lineage commitment and self-renewal with reduced carbohydrate availability leading to a maintenance of the true MuSC population a result of an increased proportions of asymmetric divisions. Finally, metabolic remodelling can be used to enhance the efficiency of MuSC transplantation.
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ItemAdipose, sex steroids and atrial arrhythmia vulnerability( 2018)Background: Pericardial adipose deposition occurs in ageing and obesity, and independently contributes to the development of atrial fibrillation. The mechanisms underlying this association are not yet understood. Investigations to date have focused on physical conduction block posed by infiltrating adipose and the secretion of pro-inflammatory/pro-fibrotic paracrine factors into the atria. Though not yet investigated in the pericardial adipose, white adipose depots are established sites of oestrogen synthesis. Considering the reported actions of oestrogens on the heart, it is hypothesised that pericardial adipose may represent an important source of local oestrogen synthesis, exerting paracrine actions on the myocardium. Research questions: 1. Do myocardial and pericardial adipose tissues express aromatase, and do locally-derived oestrogens affect the vulnerability to atrial arrhythmia? (Chapter 2) 2. Does disruption of aromatase activity in aged and obese mice influence basal cardiac electrophysiology and the susceptibility to atrial arrhythmia? (Chapter 3) 3. Can liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) sensitive methodology be used to quantify androgens and oestrogens in human and mouse myocardium and pericardial adipose tissues? (Chapter 4) Methods: Aromatase expression in human and rodent myocardium and pericardial adipose was measured by Western immunoblotting. Arrhythmia vulnerability was assessed in isolated hearts from male C57BL/6 mice (‘young’, ‘aged’ or ‘aged’ + high fat diet). Hearts were perfused with a hypokalaemic solution (2 mmol [K+]) and subjected to programmed electrical stimulation to provoke arrhythmias. In addition, hearts were perfused with acute perfusion with 17β-oestradiol (or vehicle) and arrhythmic provocation repeated. Aromatase knockout and wild type mice (male and female) were fed a control or high fat diet for 40 weeks. Mice were subjected to electrocardiographic and echocardiographic assessment prior to isolated heart atrial arrhythmia provocation experiments. Human and mouse myocardium and adipose tissues were homogenised, derivatised with dansyl chloride and subjected to LC-MS/MS for sex steroid quantification. Mass spectrometric technique was developed using the aromatase knockout as a positive control for androgens and a negative control for oestrogens. Results: 1. Aromatase is expressed in human/rodent myocardium and pericardial adipose, conferring the capacity for local androgen to oestrogen synthesis. Pericardial adipose capacity to synthesise oestrogens increased by 30-50x in aged hearts, which were significantly more vulnerable to atrial arrhythmias. (Chapter 2) 2. The aromatase knockout model of oestrogen depletion and androgen excess revealed a sex-differential phenotype in the susceptibility to atrial arrhythmia. Left atrial action potential duration was prolonged and arrhythmia vulnerability greater in female aromatase knockout mice compared to all other groups. The combined influence of extensive pericardial adipose deposition and a highly androgenic/oestrogen-depleted environment was unique to the female aromatase knockout mice and may have been decisive in driving the exacerbated vulnerability to atrial arrhythmias. (Chapter 3) 3. LC-MS/MS methodologies were optimised for the detection and quantification of sex steroids in human/mouse myocardium and adipose. Successful quantification of testosterone and progesterone was achievable, but concentrations of oestrogens in tissues were below the technical limits of detection. (Chapter 4) Conclusions: This thesis identifies that pericardial adipose expresses aromatase and indicates a probable capacity for oestrogen synthesis, hence supporting the presence of a local cardiac androgen-oestrogen system. Pericardial adipose derived oestrogens (and androgens) are recognised as probable paracrine mediators capable of altering atrial arrhythmic vulnerability. In addition, the data support the clinically observed correlation between pericardial adipose accumulation and atrial fibrillation. Mass spectrometric methodology is capable of quantifying tissue testosterone and progesterone concentrations, but tissue oestrogens are below the limits of detection. Taken together, this thesis advances the mechanistic understanding of the link between pericardial adipose accumulation and greater atrial arrhythmia vulnerability.
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ItemChronic ephedrine administration decreases brown adipose tissue activity in a randomised controlled human trial: implications for obesity( 2018)Aim Activation of Brown Adipose Tissue (BAT) may have therapeutic potential to combat obesity. Acute treatment of mice with sympathomimetic drugs activates BAT thermogenesis, and chronic treatment increases BAT thermogenic capacity. It has previously been demonstrated that human BAT is acutely responsive to oral administration of the sympathomimetic ephedrine. This study aimed to determine whether chronic treatment with ephedrine could mimic adaptive thermogenesis in humans. Methods Twenty-three healthy young men were recruited via general advertisement from Melbourne, Australia to participate in a randomised, placebo-controlled, parallel group trial. Recruited individuals were unmedicated, non-smokers, physically inactive and had no prior history of either cardiovascular disease, insulin resistance or diabetes. They were allocated to either a placebo (n=11; 22±2 years, 23±2 kg/m2) or 1.5 mg/kg/day ephedrine (active group; n=12, age 23±1 years, BMI 24±1 kg/m2) treatment group for twenty-eight days. Body composition was measured before and after the intervention by dual energy x-ray absorptiometry. BAT activity, measured before and after the twenty-eight day intervention period, via 18F-fluorodeoxyglucose positron emission tomography computed-tomography (18F-FDG PET/CT) in response to a single dose of 2.5mg/kg ephedrine, was the primary outcome measure. Results After twenty-eight days of treatment, the active treatment lost significantly more total body fat (placebo 1.1± 0.3 kg, ephedrine -0.9 ± 0.5kg; p<0.01) and visceral adipose tissue (placebo 6.4 ± 19.1g, ephedrine -134 ± 43g; p<0.01), with no change in lean mass or bone mineral content, compared with the placebo group. In response to acute ephedrine, BAT activity (change in mean standardised uptake value: placebo -3 ± 7 %, ephedrine -22 ± 6%) and the increase in systolic blood pressure were significantly reduced (p<0.05) in the active group compared with placebo. Conclusion Chronic ephedrine treatment reduced body fat content, however, it was independent of an increase in BAT activity. Rather, chronic ephedrine treatment suppressed BAT glucose disposal, suggesting that chronic ephedrine treatment decreased, rather than increased BAT activity.
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ItemInvestigating cortical oscillations, coherence and seizure susceptibility in a mouse model of autism( 2018)Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder diagnosed by difficulties in social communication, repetitive behaviour and/or restricted interests. A high proportion of ASD patients also experience seizures and abnormal brain activity as recorded via electroencephalography, however the underlying biological mechanisms of increased seizure susceptibility in ASD are unknown. Neuroligin-3 (NL3) is a neuronal adhesion protein involved in regulating synaptic structure and function. A rare point mutation at position 451 of the Neuroligin-3 amino acid sequence converts an arginine to a cysteine residue is associated with ASD and reduces NL3 protein levels by 90%. This NL3 R451C mutation was identified in two Swedish brothers with ASD, one of whom was diagnosed with comorbid epilepsy. The mutation is replicated in NL3R451C mice that exhibit reduced preference for social interactions, increased repetitive behaviours, and increased performance in memory and some motor testing. NL3R451C mice also display region-specific differences in inhibitory and excitatory neurotransmission in brain slices increased dendritic complexity, and reduced number of PV-inhibitory interneurons, all of which could contribute to changes in seizure susceptibility and oscillatory activity. Several animal models of ASD show spontaneous seizures and increased susceptibility to experimentally induced seizures, however whether NL3R451C mice have altered seizure susceptibility is unknown. It is well established that brain neuronal activity generates electrical rhythms that underlie cognitive phenomena in humans and may be altered in ASD. Gamma oscillations in the 30-100Hz range are frequently increased with demanding cognitive load and are thought to orchestrate spatially disparate cortical regions. Several clinical studies have pointed to alterations in gamma oscillations and in the coherence of oscillatory activity measured between two disparate cortical regions in ASD patients. To determine whether gamma oscillations are altered in NL3R451C mice, the glutamatergic NMDA receptor antagonist ketamine (20mg/kg; a potent inducer of gamma oscillations) was administered to adult male and female NL3R451C and WT mice and oscillatory activity recorded via electrocorticography (EcOG). PV-interneurons have been experimentally isolated to reveal they are the major drivers of gamma rhythms following ketamine administration. Baseline oscillatory activity from 6 and 10 week old male mice was examined as well as interhemispheric coherence in male and female NL3 mutants and wild type littermates. Gamma oscillatory power in male wild type and NL3R451C mice was comparable following administration of saline and was enhanced to a similar degree following ketamine (20mg/kg) administration. Non-gamma oscillations were also unchanged by ketamine administration. Interhemispheric coherence, however, was significantly higher in 6- week old male NL3R451C compared to control mice for both beta and gamma oscillation ranges. In 10-week old male mutants, there was a significant effect detected for theta, alpha and beta oscillations. Because female mice carry two copies of the X-linked NL3 gene it was hypothesised that any oscillatory activity effects may be exacerbated by the R451C mutation. Gamma band oscillations following administration of ketamine (20 mg/kg) were statistically not significant when WT, heterozygous and homozygous females were compared. Additionally, no statistically significant differences were found for interhemispheric coherence in females. To gain further understanding of the role of the NL3 R451C mutation in epilepsy, seizure susceptibility was investigated in NL3R451C male mice using pentalenetetrazole (PTZ) at both low (20 and 30 mg/kg) and high (50mg/kg) doses. Administration of high dose PTZ is associated with behavioural changes culminating to convulsive seizures. The Racine behavioural scale was used to quantify behavioural seizure severity over time in response to high dose PTZ. In mice, low dose PTZ results spike-wave discharges (SWDs) and a loss of responsiveness resembling absence seizures in patients. Electroencephalography (EcOG) recordings of neuronal activity at the cortical surface was utilised to detect SWDs following administration of low dose PTZ over a 30 minute period. NL3R451C mice showed a strong trend for shorter SWD duration although EcOG analyses of SWD frequency and duration showed similar susceptibility in both mutants and WT littermates to low dose PTZ-induced seizures. Following administration of high-dose PTZ (50mg/kg), NL3R451C mutants were slower to progress to severe seizure scores and overall progressed to milder seizures on the racine scale over the 30-minute testing period. The results of the present study show reduced seizure susceptibility and increased coherence in male NL3R451C mice. These changes might reflect underlying structural and neurochemical alterations present in NL3R451C mice. The reported increase in cortical inhibitory neurotransmission in NL3R451C mice may underlie the resistance to PTZ-induced seizures identified in this study. Similarities in gamma oscillations may indicate similar functioning of NMDA receptors and the PV-interneurons that are strongly implicated in the generation of gamma rhythms. Higher interhemispheric coherence in male NL3R451C mice could stem from differences in cognitive, behavioural and cortical changes present in this model. Further research is needed to clarify relationships between behaviour and oscillatory activity in NL3R451C mice.
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ItemMetabolic and cardiorenal adaptations to pregnancy in females born small on a high fat diet and benefits of endurance exercise training( 2017)Uteroplacental insufficiency is the major cause of intrauterine growth restriction in Western society and is often associated with adult metabolic, cardiovascular and renal diseases. Recent studies have reported that these phenotypes are exacerbated with “second hits” such as pregnancy and obesity in women born small. Pregnancy is the greatest physiological challenge facing women and, indeed, females born small are at increased risk of gestational diabetes and hypertension. Interestingly, there has been a significant increase in the number of reproductive age women who are either overweight or obese over the past few decades. Being obese or overweight is suggested to exacerbate the pregnancy complications in women born small. Importantly, exercise is reported to prevent or delay the metabolic and cardiovascular dysfunction in individuals born small. Thus, the development of targeted interventions in growth-restricted females may prevent them from developing metabolic and cardiorenal dysfunctions during pregnancy. Uteroplacental insufficiency, resulting in growth restriction, was induced by bilateral uterine vessel ligation (Restricted) or sham (Control) surgery on embryonic day 18 (E18) in Wistar-Kyoto rats. Female offspring consumed a Chow or a high fat diet (HFD; 43% kcal from fat) from 5 weeks and were mated at 20 weeks with normal male. Systolic blood pressure was measured by tail cuff in all rats prior to pregnancy (week 18). Female rats remained Sedentary or exercised on treadmills for 4 weeks before pregnancy and throughout pregnancy or only during the last two thirds of pregnancy. Rats were individually placed in an indirect open-circuit calorimeter chamber (CLAMS; 24 hours) at E16 to measure their energy expenditure and spontaneous physical activity. Systolic blood pressure was measured by tail cuff and non-fasted glucose tolerance test was performed at E18. At E19, rats were individually placed in a metabolic cage to collect urine and blood was taken by tail vein to calculate estimated glomerular filtration rate (eGFR) via measurement of urinary and plasma creatinine. Maternal plasma, pancreas, and heart were collected at E20. Fetuses and placentae were weighed and sex was confirmed using qPCR (SRY). In Chapter 3, the effect high fat feeding on the metabolic adaptations during pregnancy in rats born small was explored as was whether exercise before and during pregnancy is more beneficial in preventing these complications than exercise during pregnancy alone. Control and Restricted rats consuming a HFD were significantly heavier with more dorsal fat (+40%) and higher plasma leptin concentrations (+80%) compared to Chow-fed rats, irrespective of exercise interventions (P<0.05). Compared to Sedentary, both exercise interventions increased oxygen consumption (VO2) and respiratory exchange ratio (RER) in Restricted Chow-fed rats only (P<0.05). HFD induced glucose intolerance in Control females and exacerbated glucose intolerance in Restricted females that remained Sedentary throughout the study (P<0.05). The development and exacerbation of glucose intolerance in Control and Restricted females were prevented by exercise initiated prior to and continued during pregnancy. Furthermore, exercise before and during pregnancy increased insulin secretion in Chow-fed females and increased β-cell mass in HFD Control and Restricted females (P<0.05). No differences in spontaneous physical activity were detected across the groups. Of interest, metabolic dysfunction in Control and Restricted females was not improved by exercise initiated during pregnancy. In Chapter 4, we determined if high fat feeding exacerbates the known adverse cardiorenal adaptations to pregnancy in rats born small and whether exercise before and during pregnancy is more beneficial in preventing these complications than exercise during pregnancy alone. Sedentary Control females on a HFD altered renal function (increased eGFR; P<0.05) and this was not affected by exercise. Compared to Control, Restricted females that remained Sedentary also had an increased eGFR (P<0.05), which was not influenced by HFD or restored by both exercise interventions. No changes in pre-pregnancy systolic blood pressure were identified in all experimental groups. When pregnant, Restricted Chow- fed rats and both Control and Restricted females on a HFD had an impaired cardiovascular adaptation, with a greater reduction in systolic blood pressure during late gestation (P<0.05), and only exercise initiated before and continued during pregnancy prevented this. Additionally, Control and Restricted rats that exercised prior to and during pregnancy had an increased heart weight (normalized to tibial length) irrespective of diet at E20, indicative of physiological cardiac hypertrophy. Finally, Chapter 5 of the thesis investigated the effect of high fat feeding and endurance exercise training on the fetal outcomes in rats born small. Maternal growth restriction and HFD did not affect male and female fetal and placental weights in mothers that remained Sedentary throughout the study. Exercise initiated before and continued during pregnancy increased fetal weight in both male and female fetuses of mothers on a Chow diet, but not in mothers on a HFD. No difference in male and female fetal and placental weights were observed when mothers exercised during pregnancy only. In summary, HFD revealed and exacerbated glucose intolerance in pregnant females born of normal birth weight and born small, respectively. These were prevented by the lifestyle intervention of exercise, potentially due to improved β-cell mass. The improved glucose intolerance in Chow-fed Restricted that initiated exercise prior to pregnancy was likely contributed by increase in glucose-stimulated insulin secretion. This study also suggests that females born small have altered cardiorenal adaptations to pregnancy. Although impaired metabolic and cardiovascular adaptations during pregnancy were prevented by exercise prior to and during pregnancy, renal function was not affected by both exercise interventions. This study highlights that modifiable risk factors, such as diet and exercise, can have beneficial effects in the mother during pregnancy; particularly for females born small. By identifying females who are at high risk, especially in women born small and overweight/obese women, will ensure preventative intervention strategies to minimise adverse outcomes during pregnancy and in later life. Ultimately, these approaches may reduce the perpetuating cycle of metabolic and cardiorenal disease risk to future generations.
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ItemNeural mechanisms involved in enterotoxin- induced intestinal hypersecretion( 2017)Exotoxins of the bacteria Vibrio cholerae (cholera toxin, CT) and Clostridium difficile (C. diff., TcdA) induce rampant disease in the form of irresolvable diarrhoea causing rapid dehydration and potential death if left untreated. Both bacterial toxins affect the nervous system of the gut, the enteric nervous system (ENS), but the types of enteric neurons involved are still indistinct. Additionally, preliminary work from a collaborator showed that specific bacterial metabolites, particularly GABA produced by the microorganisms residing in the gut, exacerbate pathophysiological effects of C. diff. GABA and its receptors are expressed in several parts of the gut wall, including enteric neurons. While studies have proposed GABA to be a putative neurotransmitter in the ENS, physiological roles of GABA in the gut remain unclear. It is unknown how enterotoxins and increased GABA at the level of the gut mucosa activate underlying enteric circuitry; my PhD aimed to elucidate these mechanisms. In Chapter 3, I investigated the enteric neural pathways underlying CT effects via in vitro incubations of CT in guinea pig jejunum. Previous work highlighted the impacts of CT on secretomotor neurons; I endeavoured to expand this by examining other key neuronal subtypes. I recorded neuronal activity in the myenteric plexus (MP) up to 6 hours after CT incubation via intracellular electrophysiology. A colleague undertook similar recordings in the submucosal plexus (SMP). We found that CT induced hyperexcitability in myenteric, but not submucosal, sensory neurons. The effect was neurally mediated and required activation of NK3 tachykinin receptors, but was independent of activation of 5-HT3 receptors or NK1 tachykinin receptors, suggesting that the effects of CT on myenteric sensory neurons are likely to be indirect and via a pathway independent of 5-HT release. In Chapter 4, I determined the effects of luminal incubations TcdA and GABA on myenteric sensory neurons via electrophysiology. I found that in vitro incubations of guinea pig jejuna with TcdA or GABA also increased the excitability of myenteric sensory neurons, highlighting the key role of these neurons as a common point through which enterotoxins and GABA operate. The GABA-induced effects were inhibited by GABAB and GABAC receptor antagonists, but enhanced by a GABAA antagonist, indicating involvement of at least two distinct GABA activated pathways. The GABAA antagonist enhanced excitability on its own suggesting that tonic release of endogenous GABA may play a role in suppressing the excitability of these neurons. In Chapter 5, I explored the role of endogenous GABA in the ENS of mouse small intestine. I employed Wnt1-Cre;R26R-GCaMP3 mice, which express a fluorescent calcium indicator in the ENS, for use in Ca2+ imaging. Neurons responded to GABA exposure via activation of GABAA, GABAB and GABAC receptors in myenteric ganglia. Further, I showed that the effects of GABA were neuronal subtype specific, for example neurons immunoreactive for neuronal nitric oxide synthase rarely responded to GABA. I also demonstrated that endogenous release of GABA may inhibit activation of myenteric neurons by activation of GABAC receptors, despite such receptors exciting myenteric neurons when activated by exogenous GABA. My data also suggest that neither GABAA nor GABAB receptors contribute to synaptic transmission in this system. Further I also demonstrated the expression of GABA in neurons and varicosities surrounding specific enteric neurons within the MP. This study clarifies the complex nature of GABAergic transmission in the ENS. In Chapter 6 to further examine the effects of enterotoxins on the enteric circuitry, I made intracellular recordings from myenteric neurons following in vivo incubations of CT in mouse ileal loops. A lab member previously showed that CT increases calcium responses in the submucosal but not myenteric, neurons. In undertaking electrophysiological recordings, a striking sampling bias was revealed with exclusion of largely descending interneurons and inhibitory motor neurons being markedly underrepresented in the data set and low sampling from sensory neurons meant that significant effects in excitability may have been missed. Nevertheless in concordance with the results from Ca2+ imaging, no significant changes in excitability of myenteric neurons were found at their resting membrane potential. However, CT induced spontaneous synaptic activity in specific myenteric neurons, but the sources of this input could not be identified due to the technical difficulty of maintaining impalements, the relative rarity of myenteric sensory neurons and the sampling bias. The data suggest a minor role for myenteric neurons in CT-induced hypersecretion in vivo. In Chapter 7 I employed the high throughput assay of Ca2+ imaging to perform a more extensive examination of the effects of TcdA on the ENS. I utilized the well-established ileal loop mouse model and incubated TcdA in vivo. Spontaneous and neurally-stimulated calcium responses were reduced in submucosal neurons, and myenteric neuronal activity was unchanged. However enteric neurons in regions of the gastrointestinal tract off-target from the site of acute toxin exposure were activated during the incubation as indicated by expression of activity dependent markers. This off target effect could possibly be due to release of inflammatory cytokines into the circulation or extrinsic neural pathways. In all, I have demonstrated a generality in the actions of enterotoxins and GABA as the pathways they activate converge to excite myenteric sensory neurons which may lead to activation of submucosal secretomotor neurons. I have extended our understanding of the role of GABA in the ENS as a means to elucidate the mechanisms through which microbial metabolites act and contribute to disease. Using the mouse ileal loop model, I further defined the effects of enterotoxins on the enteric circuitry. In this way, my thesis highlights neural elements involved in the mechanisms underlying enterotoxin-induced hypersecretion and identifies potential avenues for future research.
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ItemPathology of glycogen excess in diabetic cardiomyopathy( 2017)Background: Diabetic cardiomyopathy is a distinct cardiac pathology and the underlying mechanisms are unknown. Elevated glycogen content has been observed in the diabetic human myocardium, first recorded 80 years ago, suggesting that despite impaired glucose uptake cardiomyocytes accumulate glycogen. Anecdotal evidence of glycogen accumulation in the diabetic myocardium has since been recorded in the literature but a systematic investigation of this paradoxical phenomenon has not been conducted. Glycogen storage diseases demonstrate that increased cardiac glycogen is associated with severe functional deficits, and therefore the observed glycogen ‘excess’ in diabetic hearts may be an important and novel agent of pathology in diabetic cardiomyopathy. Aim: This body of work aimed to systematically investigate the role myocardial glycogen accumulation in diabetic cardiomyopathy, with a focus on glycophagy, a glycogen-specific autophagy process. Key metabolic signaling pathways (insulin, AMPK, β-adrenergic) were interrogated to investigate their therapeutic potential. The four experimental questions addressed in this thesis are: 1. Does myocardial glycogen accumulation contribute to functional deficits in the diabetic heart? (Chapter 2) 2. What glycogen processing mechanisms are disrupted and may be associated with glycogen accumulation in the diabetic myocardium? (Chapter 2) 3. Do simulated hyperglycemic and hyperinsulinemic conditions mediate cardiomyocyte glycogen accumulation? (Chapter 3) 4. Can key metabolic signaling pathways (AMPK, β-adrenergic signaling) be exploited to degrade excess cardiomyocyte glycogen? (Chapter 4) Methods: Type 1 diabetes (T1D) was induced in male Sprague-Dawley rats using Streptozotocin. C57Bl/6 mice were fed a high fat diet to induce obesity and insulin resistance – a state of early type 2 diabetes (T2D). Human atrial tissue from diabetic patients were examined for glycogen content. Echocardiography was conducted to assess functional outcomes in diabetic animals. Neonatal rat ventricular cardiomyocytes were cultured in extracellular high glucose (30mM) and insulin (1nM) and/or had suppressed GABARAPL1 expression (siRNA, siGABARAPL1). Influence of β-adrenergic or AMPK activation was assessed using isoproterenol (100µM, 1 hour) or AICAR (30µM, 30 minutes), respectively. Glycogen content in cardiac tissue homogenates and cell lysates was measured via enzymatic assay. Molecular markers of key signaling pathways were investigated using immunoblotting, immunohistochemistry and qPCR. Results: Some of the overall findings of this investigation are that: 1. Myocardial glycogen accumulation in in vivo models of insulin resistance and progressed T1D is associated with diastolic and systolic dysfunction. 2. Myocardial glycogen accumulation is associated with decreased GABARAPL1 lipidation, suggesting a disruption in glycophagosome scaffold processing in the insulin resistant mouse and diabetic human myocardium. This finding was also established in vitro where a suppression of GABARAPL1 mRNA induced cardiomyocyte glycogen excess. 3. High extracellular glucose (simulated hyperglycemia) only increases cardiomyocyte glycogen content in the presence of insulin and is associated with increased expression levels of the glycophagy adapter protein STBD1 in vitro. 4. In vitro activation of β-adrenergic signaling mediates a reduction in cardiomyocyte glycogen via activation of glycogen phosphorylase when glycophagy is disrupted (siGABARAPL1). In vitro activation of AMPK signaling decreases cardiomyocyte glycogen induced by disrupted glycophagy (siGABARAPL1), but is not effective in modulating glycogen loading induced by high extracellular glucose (simulated hyperglycemia). This study identifies glycogen accumulation as a novel agent of pathology in the development of diabetic cardiomyopathy, associated with a disruption in glycophagy. It is the first to show that cardiac dysfunction is linked with myocardial glycogen accumulation. In a glycophagy compromised setting, AMPK and β-adrenergic signaling may provide potential therapeutic targets to rescue cardiac glycogen excess. An increased understanding of the complex signaling pathways mediating glycogen synthesis and storage in early diabetes may provide a platform for the development of cardiac specific, targeted therapeutic interventions in diabetes.
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