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.