Physiology - Theses
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ItemCharacterisation of synaptic transmission and angiotensin II type 1A receptor activity at nucleus of the solitary tract neurons( 2017)The ability to sense changes in the body and external environment, and to undertake appropriate physiological responses is essential for survival. Cranial visceral afferents continuously monitor the state of visceral organs and relay this to the central nervous system (CNS). The CNS integrates this input which results in neuroendocrine and autonomic nervous system activity to maintain homeostasis. Visceral cranial nerves first synapse onto neurons in the CNS in the nucleus of the solitary tract (NTS). These inputs are high probability release sites that strongly affect the activity of NTS neurons. This allows high fidelity relay of visceral signals to effector nuclei around the CNS and recruitment of appropriate responses. These reflexes are believed to be gated at the NTS by a variety of mechanisms. This thesis focussed on two systems, the renin-angiotensin system (RAS) and the hypothalamus, both of which are known to play important roles in modulating NTS activity in health and disease. The cellular mechanisms by which the RAS and input from the hypothalamus directly affect NTS neurons are unknown. Modulation of reflexes play an important role in health and disease, and these studies strengthen our understanding of visceral sensory relay in the NTS. It is well established that microinjection of angiotensin II (AngII), the main effector peptide of the RAS, into the NTS modulates peripheral baroreceptor and chemoreceptor reflexes via the angiotensin II type 1A receptor (AT1AR). Furthermore, in vitro intracellular electrophysiological recordings in the NTS have revealed a subset of neurons that are depolarised by AngII application. However, the AT1AR is expressed in neuronal cell bodies, visceral afferent terminals, glia, and endothelial cells of the NTS, and the effects of AngII specifically on AT1AR-expressing neurons are unknown. Chapter 3 aimed to determine the effect of AngII application on membrane potential (VM) and voltage-gated ion channel activity in AT1AR-expressing neurons within the NTS. Experiments were performed using transgenic mice whereby expression of green fluorescent protein (GFP) was driven by the AT1AR-promoter (AT1AR-GFP). NTS neurons expressing GFP were identified in coronal slices (250 μm) of medulla oblongata and whole-cell electrophysiological recordings were performed to examine passive membrane properties, voltage-activated currents, excitatory post-synaptic potentials (EPSPs) and VM in this neuronal sub-population. Activity was recorded from a total of 53 neurons before and during bath application of AngII, either alone or in combination with the following: AT1R antagonist candesartan (Cand), AT2R antagonist PD123319 (PD), or voltage-gated sodium channel inhibitor tetrodotoxin (TTX). The application of AngII increased VM by between 0.9 and 11.3 mV with an average increase of 3 mV. This effect was blocked by Cand but not PD. TTX did not prevent AngII-induced increases in VM and there was no effect of AngII in the frequency or amplitude of EPSPs. This indicates the AngII-dependent increase in VM was dependent on AT1AR activity changing the ionic membrane properties of the recorded neuron. To determine the ionic current responsible for this change in VM, the expression and kinetics of voltage-activated currents before and after AT1AR activation was assessed. There were no changes in input resistance or the magnitude of several voltage-dependent potassium currents (IA, IK, or IM) in response to AngII application. This indicates that other currents are responsible for the AngII-induced increase in VM, with a change in inward cation currents, such as voltage-dependent calcium currents, likely. The centrally-projecting axons of visceral afferents are carried to the NTS by the solitary tract (ST). The NTS neurons which receive ST input directly are termed second-order, while those that receive indirect input via NTS interneurons are termed higher-order. Inputs from the ST are heterogeneous, consisting of either fast conducting Aδ-fibres or slow conducting C-fibres. Aδ- and C-fibre afferents relay low threshold high frequency input and high threshold low frequency input, respectively. The proportion of AT1AR-expressing NTS neurons that are second-order, and whether this sub-population receive Aδ- or C-fibre input, is unknown. The application of AngII has been shown to potentiate excitatory ST input to a sub-population of NTS neurons. However, it is unknown whether this sub-population is comprised of AT1AR-expressing neurons. This knowledge will enhance our understanding of the role of AT1AR-expressing NTS neurons in sensory relay and reveal an alternative pathway that AngII modulates sensory input. Chapter 4 aimed to determine what proportion of AT1AR-expressing neurons, if any, are second-order, and what proportion of these are C-fibre ST input. Furthermore, the effect of AngII on ST input to second-order AT1AR-expressing neurons was investigated. Horizontal slices of medulla oblongata (250 μm) containing the ST and NTS were prepared from AT1AR-GFP mice. Whole-cell electrophysiological recordings were made from 56 AT1AR-expressing neurons during the electrical recruitment of ST afferents. Seventy per cent of these neurons received direct ST input as indicated by low temporal variability of excitatory input following repeated ST excitation. The majority of C-fibres express transient receptor potential, vallinoid type 1 (TRPV1), making them sensitive to capsaicin (CAP). A subset of eight second-order AT1AR-expressing neurons was assessed for fibre type by the application of CAP. In five of eight neurons, ST input was reduced by CAP with a concurrent increase in spontaneous activity, indicating AT1AR-expressing neurons predominantly receive C-fibre input. Finally, AngII was applied to a subset of eleven second-order AT1AR-expressing neurons during recruitment of ST input and no changes were observed. This chapter revealed AT1AR-expressing NTS neurons predominantly receive direct C-fibre input, and input from the ST to these neurons was not potentiated by AngII. Neurons of the paraventricular nucleus of the hypothalamus (PVN) send projections to the NTS and thus have the potential to gate visceral sensory input. Some of these PVN neurons provide peptidergic inputs to the NTS, potentially including an angiotensinergic projection. However, the main neurotransmitters involved in this projection and whether the PVN makes functional synapses directly to NTS neurons remain unknown. Chapter 5 aimed to determine the nature of this connection including; the neurotransmitters involved, whether there is selective targeting of AT1AR-expressing NTS neurons and what effect convergent ST and PVN input has on NTS neuronal excitability. To address these aims, a replication-incompetent adeno-associated virus was microinjected into the region of the PVN of 35 adult AT1AR-GFP mice. Viral infection caused expression of a light-gated cation channel, humanised channel rhodopsin 2 (ChR2) with a point mutation designed to increase membrane trafficking (H134R). The ChR2 sequence was fused to a fluorescent reporter (tdTomato) and the transgene expressed under the control of a ubiquitous promotor. After three months, a 250 μm horizontal slice of the medulla oblongata was produced, including the ST and NTS, and whole-cell electrophysiological recordings were made. An LED (450 nm wavelength) was used to stimulate ChR2, thus activating the axonal terminals of transduced hypothalamic neurons in the NTS. Electrical stimulation of the ST was performed to characterise the NTS neuron. The pipette solution contained 0.5% biocytin and, after recordings, slices were fixed in 4% paraformaldehyde and immunohistochemistry was performed to visualise PVN inputs and recorded NTS neurons. Fourteen animals showed evidence of LED synchronised post-synaptic currents to at least one recorded NTS neuron. Of the 86 neurons recorded, LED-induced EPSCs were evident in 21 neurons while LED-induced IPSCs were recorded at two neurons. A single neuron received both LED-induced EPSCs and IPSCs. The remaining 62 NTS neurons recorded from successfully transduced animals did not receive input from the hypothalamus. Excitatory hypothalamic input showed small amplitudes and variable temporal relationship to stimulation. The response was mediated by the release of glutamate acting at fast ionotropic receptors. The EPSCs displayed frequency-dependent depression indicating that this input is a high probability release site. In a subset of nine of NTS neurons receiving EPSCs, the application of TTX blocked the synaptic input. In three cases, the response returned with the co-application of TTX and the voltage-gated K+ channel inhibitor, 4-aminopyrimidine (4AP). This suggests that the majority of excitatory hypothalamic input (six of nine neurons) was indirect, arriving via an NTS interneuron. Immunofluorescent confocal microscopy showed that an NTS neuron confirmed to receive a direct excitatory hypothalamic input had little apposition between tdTomato-expressing terminals and the neurons soma. This may indicate selective dendritic targeting of hypothalamic inputs. Hypothalamic IPSCs were mostly inhibited by the application of an excitatory fast ionotropic receptor antagonist, indicating that these were also indirect, and dependent on PVN excitatory input to an inhibitory NTS interneuron. Fourteen of 22 NTS neurons receiving hypothalamic input also expressed GFP. Convergent input from LED and ST stimulation resulted in the superimposition of currents. During recordings of VM, concurrent hypothalamic stimulation increased the likelihood of observing an action potential during ST stimulation. This indicates that hypothalamic input to NTS neurons is predominantly excitatory and can result in both the recruitment of local excitatory and inhibitory NTS interneurons. Sixty-three per cent of neurons receiving hypothalamic input expressed the AT1AR and potentiated sensory relay in the NTS. These results provide a greater understanding of the mechanisms by which the RAS and descending hypothalamic projections gate viscerosensory input from the viscera. AngII increased the VM of a sub-population of NTS neurons via activation of the AT1AR. Given that the majority of AT1AR-expressing neurons were second-order, receptor activation may play an integral part in modulating the relay of visceral input to the CNS via C-fibres and, to a lesser extent, Aδ-fibres. Likewise, hypothalamic projections to the NTS have the potential to increase the propagation of ST input, causing action potentials in NTS neurons. This can result in local excitatory or inhibitory NTS interneuron recruitment. These data suggest an important role for AT1AR-expressing neurons in reflex relay in the NTS, and elucidates signalling and synaptic mechanisms by which reflexes can be modulated.
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ItemDiabetic cardiomyopathy: sex-specific aspects of functional, structural and molecular remodelling( 2017)Background: Clinical studies have revealed increased cardiovascular risk in diabetic patients, which is substantially elevated in women. Perplexingly, while there has been extensive experimental effort in characterising cardiac dysfunction in the progression of diabetic cardiomyopathy, studies investigating sex differences are limited. A mechanistic understanding of sexual dimorphism in diabetic cardiomyopathy remains to be achieved. Aim: The aim of this Thesis was to examine female susceptibility to cardiac pathology in type 1 diabetes (T1D). In particular, this thesis focussed on examining cardiac responses to diabetes (functional and molecular) under basal conditions, during ischemia and with increased cardiac renin angiotensin system (RAS) signalling. The four experimental questions addressed in this thesis are: 1. Are systemic and cardiac T1D phenotypes different between males and females? [Chapter 3] 2. Is there an accentuated female vulnerability to ischemia reperfusion injury in T1D? [Chapter 4] 3. Are there sex-specific changes in cell death, autophagy and metabolism associated with diabetes? [Chapter 5] 4. Does cardiac RAS upregulation interact with sex-specific cardiac responses in T1D? [Chapter 6] Methods: A wide range of in vivo, ex vivo and molecular strategies were employed to characterise the role of sex differences in a streptozotocin (STZ)-induced T1D mouse model. Echocardiographic assessment was performed to examine T1D-induced functional and structural deficits in vivo. Ex vivo isolated heart perfusion analysis was used to characterise the role of sex differences in ischemia-reperfusion injury and recovery in T1D. The mechanistic basis of T1D-induced cardiac pathology was evaluated with various histological, biochemical and molecular techniques. Molecular findings from T1D models were also compared against changes from type 2 diabetic (T2D) mouse models (lean and obese). Finally, the role of RAS in exacerbating the T1D phenotype was assessed using a cardiac-specific angiotensinogen overexpressing mouse model. Results: The overall findings of this thesis are: 1. Although the extent of hyperglycaemia and increase in glycated haemoglobin (HbA1c) was less marked in female T1D in comparison to male T1D, diastolic dysfunction was evident in female T1D, but not in male T1D mice. 2. In males, diabetic hearts showed greater reperfusion recovery associated with reduced cardiac glycogen levels post-ischemia, suggesting better glycogen utilisation during ischemia, compared to male controls. In contrast, despite an earlier onset of ischemic contracture, the reperfusion recovery and glycogen levels were unchanged in female T1D hearts, compared to female control hearts. 3. GABARAPL1, a gene responsible for lysosomal breakdown of glycogen, was upregulated in T1D male hearts, whereas genes from conventional glycogen breakdown pathways (glycogen phosphorylase and glycogen debranching enzyme) were increased in female T1D. In addition, a pronounced increase in expression of genes from macro-autophagy pathway (protein bulk degradation) and apoptotic cell death pathway genes were observed in female T1D but not male T1D hearts. Interestingly, in lean and obese T2D mice, contrasting cardiac gene expression responses were observed in glycogen metabolic and macro-autophagy pathways. 4. With elevated cardiac AngII, T1D-induced cardiac functional and structural changes were exacerbated in males, but these changes were not apparent in females. Conclusion: Collectively, the novel findings in this thesis have contributed new knowledge to the literature on sex-specific attributes of diabetic cardiomyopathy. This study is the first demonstration that a less pronounced hyperglycaemic response in T1D female mice is associated with more marked functional cardiac pathology. This female vulnerability may be partially attributed to a preferential slower/inefficient processing of glycogen and heightened cell death pathology, evidenced from pronounced autophagic drive in female T1D mice. A sex-specific role for cardiac RAS in exacerbating the T1D phenotype has also been identified. The findings in this thesis support a case for sex-specific progression of diabetic cardiac pathology.
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ItemIdentification of the phenotype of the angiotensin II type 1A receptor-expressing neurons involved in modulating the baroreceptor-heart rate reflex( 2016)Blood pressure (BP) regulation requires a complex interaction between different physiological systems to ensure adequate tissue blood flow under different physiological conditions. One of these is the baroreceptor reflex which modulates heart rate (HR) and total peripheral resistance (TPR) to modulate BP homeostasis. Circulating angiotensin II (ANG II) acts via its type 1 receptor (AT1R) to attenuate the barorereceptor-heart rate (HR) response. It is currently unknown which AT1R –expressing cell type mediates this modulatory effect of ANG II on the baroreflex. Thus, overarching aim of this thesis is to investigate which cell types, involved in the modulation of the baroreceptor reflex express the AT1R. To achieve this aim, I used a combination of genetic tools to remove AT1Rs from specific cell populations. In chapter three, the baroreceptor-HR response was investigated in the global AT1AR knockout (AT1AR-KO) and wildtype (AT1AR-WT) mice under artificial ventilation and isoflurane anaesthesia. Baroreceptors were activated by changes in blood pressure induced by bolus injections or slow infusions of phenylephrine (PhE) or sodium nitroprusside (SNP) to increase or decrease the blood pressure respectively. No impairment in the baroreceptor-HR response was observed in AT1AR-KO mice. In chapter four, I have attempted to generate a brain-specific knock out of the AT1ARs using the conditional, CRE-recombinase-mediated deletion. Unfortunately, I was unable to demonstrate any obvious reduction in the expression of AT1ARs in the brains of these mice. The baroreceptor-HR response was intact in these animals. An abberant tachycardiac response to systemic ANG II was observed, but the cellular basis for this could not be determined. The nucleus of the solitary tract (NTS) is the first synapse of the baroreceptor afferent neurons. It has a high density of AT1Rs and is invovlved in modulation of baroreceptor-HR response by ANG II. However, little is known about the properties of the AT1R-expressing neurons in the NTS. Chapter five addresses this issue using a transgenic mouse where the AT1AR- promoter drives GFP expression. AT1AR is widely expressed in many subregions of the NTS in cells resembling neurons. Co-expression of the AT1AR-GFP and either Phox2b or tyrosine hydroxylase (TH), occurred in a distinct pattern in different subregions of the NTS. This suggests that AT1R-expressing neurons in the NTS are a heterogenous population with a complexity that reflects the diverse function of ANG II in the NTS. In chapter six, the role of subpopulations of AT1R-expressing cells in the NTS in modulating the sensitivity of the baroreceptor-HR responses was investigated. I observed that the sensitivity was significantly enhanced in mice in which the AT1AR was deleted from Phox2-expressing neurons using recombinant viral approaches, but was un altered in transgenic mice with AT1AR deletion from TH-expressing neurons. This suggests that AT1ARs on TH-, Phox2+ cells tonically inhibit the baroreceptor-HR response in adult mice. The PhE induced baroreceptor-HR responses was significantly enhanced in AT1AR-FL mice that received bilateral injections of Lv-PRSx8-CRE in the NTS but was unaltered in the ThCRE+ x AT1AR-FL mice. This suggested that AT1ARs on TH-, Phox2+ cells tonically inhibits the baroreceptor-HR responses in adult mice. In conclusion, we have found that the AT1R-expressing cells in the NTS are a heterogeneous population of cells. Amongst these, the AT1AR on TH-, Phox2+ cells attenuate the baroreceptor-HR response. Further investigation is required to fully understand the functional properties of other AT1R-expressing cells in the NTS.
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ItemCharacterization of mechanisms of myocardial remodeling in genetic models of cardiac hypertrophy( 2005-12)Cardiac hypertrophy is clinically defined as a relative increase in heart size associated with a thickening of the ventricular wall. It is a common feature of individuals suffering from different cardio-vascular or metabolic conditions and leads to heart failure. The structural, functional and molecular mechanisms which induce hypertrophy independent of hemodynamic alterations are poorly characterized. In this study, questions about whether cardiac-specific neuro-endocrine activation or metabolic imbalance are sufficient to induce hypertrophic structural and functional remodeling are addressed using genetically manipulated mouse models of primary cardiac hypertrophy. (For complete abstract open document)
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ItemThe role of angiotensinogen in the brain renin-angiotensin system( 2011)The renin-angiotensin system (RAS) potently regulates blood pressure and fluid homeostasis. Athough originally described as circulating, humoral system, it is made more complex by the existence of several, potentially independent, tissue RAS. The brain RAS is the focus of the present PhD thesis and serendipitously, this year marks the 40th anniversary of its purported discovery by Fischer-Ferrario and Ganten. During this time, each component of the RAS has been identified within the brain, including enzymes of the extended RAS cascade such as angiotensin-converting enzyme type 2 and the mas receptor. The only known precursor of angiotensin peptides is angiotensinogen (AGT) and in the brain, it is produced predominantly by astrocytes. Physiological studies have shown that the predominant actions of angiotensin II (Ang II) in the brain involve cardiovascular regulation, consistent the systemic RAS. These studies have led to the hypothesis that the brain RAS functions and is regulated independently of the peripheral system. The first aim of this thesis was to determine whether Ang II modulates AGT production by astrocytes. Initial experiments were performed on primary astrocyte cultures from neonatal mice. Surprisingly, despite rigorous analyses, an Ang II binding site could not be detected in these cultures. Preliminary data from a transgenic mouse expressing a fluorescent protein via the angiotensin type 1A receptor (AT1AR) promoter did not reveal astrocytic expression of the receptor under basal conditions. Consistent with the absence of AT1ARs, AGT gene expression was not altered by incubation of cultured astrocytes with Ang II. However, when cultured astrocytes were transduced with AT1ARs they exhibited a profound decrease in AGT gene expression after 24 hours (91.4 % ± 1.8 %, P<0.01, n=4). To confirm this observation in vivo, recombinant adenoviral vectors were used to express the wild type or a constitutively active mutant (N111G) version of the AT1AR in rat astrocytes. A marked reduction in AGT immunoreactivity and gene expression was observed in astrocytes that expressed the constitutively active AT1AR, but not in astrocytes expressing the wild type receptor or control vector. Together, the studies described in Chapters two and three suggest a role for G-protein coupled receptor-mediated intracellular signalling pathways in the modulation of astrocytic AGT. Whilst all components of the RAS have been identified in the brain, their global distribution and subcellular location do not provide an intuitive model for Ang biosynthesis. In particular, it is not clear whether astrocytic AGT is required for local Ang II formation and activation of angiotensin receptors. Current methods are not adequate to resolve the relative importance and anatomy of the brain RAS in vivo without confounding influences by the systemic RAS. To address this issue, new technology incorporating novel RNA interference technology, was used to knockdown endogenous AGT gene expression specifically from astrocytes within a single brain region. Of the three microRNAs designed to knockdown AGT expression, the most efficient (miR930) was selected for use in vivo. A recombinant adenovirus was synthesised to encode miR930 and a reporter gene driven by a ubiquitous promoter. Substantial knockdown of AGT expression was observed seven days after microinjection of this construct into the rat brain nucleus of the solitary tract. However, AGT was also decreased following transduction with a negative-control microRNA produced in parallel with miR930, which has no homology to any known gene. This non-specific effect of the control microRNA was not observed in cultured cells transiently transfected with AGT. Therefore, it was reasoned that the recombinant adenoviral vector or foreign promoter were causing this non-specific AGT knockdown. Since the level of AGT knockdown induced by the control microRNA was as extensive as that of miR930, it was reasonable to conclude that a proportion of the knockdown achieved by this method was specific. Therefore, another vector was made to deliver the miR930 and its control in vivo. Recombinant lentiviruses were used, since they were considered less immunogenic. To direct expression in astrocytes, a Mokola lyssavirus protein coat was used and transgenes were driven by an astrocyte-specific promoter. Fourteen days after administration of this particular vector into the brain, robust AGT knockdown was observed. Unfortunately, the control microRNA also caused a robust decrease in AGT expression. Other astrocyte proteins were also affected by transduction with recombinant lentiviruses, somewhat consistent with the profile of activated astrocytes during an immune response. These non-specific effects could be attributed to a number of issues: including local mechanical perturbation; over-expression of transgenes; contaminants in the viral suspension; or local responses to viral infection. Further improvements in this technology and optimisation strategies will enable specific AGT knockdown in astrocytes in vivo and help resolve the organisation of this complex system in the brain.