published_or_final_version / Physiology / Master / Master of Philosophy

Adiponectin regulates feeding behavior, energy expenditure and autonomic function through the activation of two receptors present in nuclei throughout the central nervous system, however much remains unknown about the mechanisms mediating these effects. Here I investigate the actions of adiponectin in autonomic centers of the hypothalamus (the paraventricular nucleus) and brainstem (the nucleus of the solitary tract) through examining molecular, electrical, hormonal and physiological consequences of peptidergic signalling. RT-PCR and in situ hybridization experiments demonstrate the presence of AdipoR1 and AdipoR2 mRNA in the paraventricular nucleus. Investigation of the electrical consequences following receptor activation in the paraventricular nucleus indicates that magnocellular-oxytocin cells are homogeneously inhibited while magnocellular-vasopressin neurons display mixed responses. Single cell RT-PCR analysis shows oxytocin neurons express both receptors while vasopressin neurons express either both receptors or one receptor. Co-expressing oxytocin and vasopressin neurons express neither receptor and are not affected by adiponectin. Median eminence projecting corticotropin releasing hormone neurons, brainstem projecting oxytocin neurons, and thyrotropin releasing hormone neurons are all depolarized by adiponectin. Plasma adrenocorticotropin hormone concentration is increased following intracerebroventricular injections of adiponectin. I demonstrate that the nucleus of the solitary tract, the primary cardiovascular regulation site of the medulla, expresses mRNA for AdipoR1 and AdipoR2 and mediates adiponectin induced hypotension. Adiponectin has electrical effects on a majority of medial solitary tract neurons and depolarizes those expressing mRNA for the hypotensive neuropeptide Y, revealing a central mechanism to modulate blood pressure. Finally, I show that adiponectin controls paraventricular nucleus neuron excitability by either inhibiting a tetraethyl ammonium-sensitive potassium current thereby depolarizing neurons or activating a glibenclamide-sensitive voltage independent potassium current hyperpolarizing neurons. Therefore, adiponectin differentially modulates potassium current to confer its central effects. These results are the first to show the physiological and electrical actions of adiponectin on individual neurons in blood brain barrier protected central autonomic nuclei. This thesis provides a framework for how adiponectin acts centrally to coordinate whole body energy homeostasis and feeding behavior in the rat. / Thesis (Ph.D, Physiology) -- Queen's University, 2009-09-15 16:50:13.933

Thesis (M. Phil.)--University of Hong Kong, 1995. / Includes bibliographical references (leaf 84-110).

OBJECTIVE: Previous studies have shown the significant contribution of sympathoinhibition in response to sodium loading to prevent increases in mean arterial blood pressure in salt resistant phenotypes. It has also been shown that brain Gαi2 protein gated signal transduction plays a major role in this pathway, however, the specific mechanisms through which this pathway is activated remain less well understood. The purpose of this study was to elucidate the relative contribution of increased sodium in either the plasma or the cerebrospinal fluid (CSF) to the regulation of mean arterial pressure and natriuresis. Additionally we explored the potential for using the novel CLARITY Imaging technique to identify the relative activity of neurons in areas of the brain thought to play a major role in body fluid homeostasis in response to salt. METHODS: Rats that were pre-treated with either scrambled or Gαi2 oligodeoxynucleotides (ODN), to selectively down regulate brain Gαi2 proteins, were challenged either peripherally or centrally with sodium. Upon sodium loading physiological parameters were measured for two hours after which the animal's brains were recovered for immunohistochemical (IHC) analysis of the paraventricular nucleus, a known regulatory center for body fluid homeostasis and blood pressure regulation. Additionally we adapted a version of the published CLARITY Imaging protocols for optically clearing tissue through application of electrophoretic tissue clearing (ETC) to a larger rat model. RESULTS: In scrambled ODN pre-treated rats we observed a temporary increase in MAP in response to both the peripheral and central sodium challenge. In the Gαi2 ODN pre-treated animals we saw some form of attenuation to this response in both studies, however, where in the peripheral challenge there was an increase in the amount of time that it took the rats to return to normotension with no alteration in natriuresis, in the central challenge there was a large attenuation in natriuresis with no differences in the time to return to baseline MAP. Our IHC analysis also showed a decrease in neuronal activation of paraventricular medial parvocellular neurons in Gαi2 pre-treated rats that were challenged peripherally vs their SCR pre-treated counterparts. No such difference was observed in either of the pre-treatment groups from the central sodium challenge study. In the CLARITY study we found that it is possible to adapt the method for optically clearing tissue to the larger model, however, we encountered several issues related to tissue swelling and peripheral tissue damage. CONCLUSION: Based on our current results it seems evident that there are at least two different mechanisms that activate the cardiovascular regulatory control centers in the brain that prevent long term increases in mean arterial pressure in response to increased salt. It also appears that these two different mechanisms are triggered either by increases in plasma or CSF salinity, though which of these two mechanisms may be directly responsible for the development of salt sensitive hypertension requires further investigation. While we had some success at optically clearing larger tissue volumes through ETC, problems we encountered with maintaining tissue integrity for investigations of intact neural networks prevented us from applying this technique, in its current form, to our investigation of salt sensitive hypertension.

Beacon is a 73-amino acid peptide encoded by a novel gene in the hypothalamus of Israeli sand rat Psammomys obesus. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical techniques were used to investigate the presence of beacon mRNA and the distribution of beacon-immunoreactivity (irBC) in the hypothalamus of ICR mice. RT-PCR experiments revealed beacon mRNA in the mouse hypothalamus. Using a rabbit polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73), irBC was detected in the mouse hypothalamus and pituitary. In the hypothalamus, irBC was concentrated in perikarya of the supraoptic (SO), paraventricular (PVH) and accessory neurosecretory nuclei and in cell processes of the median eminence and pituitary stalk. In the pituitary, irBC was noted mainly in the posterior lobe. Double-labeling the hypothalamic sections with guinea-pig vasopressin-antiserum or mouse monoclonal oxytocin-antibody and beacon-antiserum revealed that <30% of vasopressin-immunoreactive neurons and nearly all oxytocin-immunoreactive neurons in the PVH and SO were irBC. The result shows the presence of beacon mRNA in the mouse hypothalamus, and the distribution of irBC is distinctively different from that reported in the hypothalamus of Psammomys obesus, but similar to that of the Sprague-Dawley rats described in our earlier study. More interestingly, Blast search uncovered a 73-amino acid peptide, human ubiquitin-like 5, which has the same exact sequence as beacon. Thus, irBC observed in the mouse brain could be that of ubiquitin-like 5.

With the use of an antiserum against human apelin-36, apelin-immunoreactivity (irAP) was detected in neurons and cell processes of the supraoptic nucleus (SO), paraventricular nucleus (PVH), accessory neurosecretory nuclei (Acc) and suprachiasmatic nucleus. Strongly labeled cells/processes were noted in the internal layer of the median eminence, infundibular stem, anterior and posterior pituitary. Double-labeling the sections with goat polyclonal neurophysin I-antiserum and rabbit polyclonal apelin-antiserum revealed a population of magnocellular neurons in the PVH, SO and Acc expressing both irAP and neurophysin I-immunoreactivity (irNP), the latter being a marker of oxytocin-containing neurons. By inference, the AP-positive but irNP-negative magnocellular neurons could be vasopressin-containing. The presence of irAP in certain hypothalamic nuclei and pituitary suggests that the peptide may be a signaling molecule released from the hypothalamic-hypophysial axis.

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No description available.

Inflammatory Bowel Disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract, often associated with reduced food intake (anorexia) and weight loss. The anorexia manifest following gastrointestinal inflammation can only be expressed if appropriate signals are communicated from the inflamed segment to the brain. Yet, the nature of these signals, and the identity of the brain sites processing these anorexigenic signals, are unknown. The present experiment evaluates the contribution of the paraventricular nucleus (PVN), a brain site rich in corticotropin releasing factor (CRF) receptors and known to be involved in the control of food intake, in the anorexia associated with experimental colitis. Colitis was induced, by trinitrobenzenesulfonic acid (TNB) treatment, in animals in which the PVN was ablated or in rats with sham brain surgeries. Results indicated clearly that the expression of the anorexia following TNB treatment is fully expressed even in the absence of the PVN. This result indicates that the integrity of the PVN is not necessary for the reduction of eating associated with intestinal inflammation, thus suggesting that CRF is also not critical to colitis-induced anorexia. inflammatory bowel disease (IBD); feeding; anorexia; gut-brain communication; paraventricular nucleus (PVN); interleukin-1 (IL-l); corticotropin-releasing-factor (CRF); neuroimmunology / Thesis / Master of Science (MS)

A redução na pressão parcial de O2 é detectada pelos quimiorreceptores periféricos que sinalizam ao sistema nervoso central para que haja uma correção na homeostasia. Estudos neuroanatômicos mostram que neurônios C1 enviam projeções para núcleo paraventricular do hipotálamo (PVH), mas, pouco descrevem a participação desta via em uma situação de hipóxia. Ademais, o envolvimento de mecanismos neuroimunes no controle neural cardiorrespiratório durante a hipóxia não está esclarecido. Neste estudo mostramos que neurônios catecolaminérgicos do BVLr/C1 ativados por hipóxia se projetam para o PVH, e que a integridade destes neurônios é essencial para que neurônios do PVH sejam ativados por hipóxia. Além disso, o tratamento com minociclina alterou a expressão de mediadores inflamatórios no BVLr e PVH, a expressão de Fos e as repostas respiratória e autônoma desencadeadas pela hipóxia. Estes resultados conferem uma importante caracterização sobre a distribuição dos neurônios catecolaminérgicos do BVLr/C1 que são ativados por hipóxia e se projetam para o PVH. Além de mostrar que a hipóxia pode desencadear mecanismos neuroimunes que possivelmente envolvem a participação da microglia e também recrutam a via neural C1- PVH. / The reduction in the O2 partial pressure is detected by the peripheral chemoreceptors that send information to central nervous system to correct the homeostasis. Neuroanatomical studies show that C1 neurons send projections to the paraventricular hypothalamic nucleus (PVH), but rather describe the involvement of this pathway in a hypoxic situation. Furthermore, the potential involvement of neuroimmune mechanisms in cardiorespiratory neural control during hypoxia is unclear. In this study we show that catecholaminergic neurons localized in rostral ventrolateral medulla (RVLM) / C1 cells activated by hypoxia send projections to the PVH, and the integrity of these neurons is essential for PVH neurons be activated by hypoxia. Moreover, treatment with Minocycline changed the expression of inflammatory mediators in RVLM and PVH, the expression of Fos in these nucleus, and respiratory and autonomic responses elicited by hypoxia. These findings provide an important characterization of the distribution of catecholaminergic RVLM / C1 neurons that are activated by hypoxia and project to the PVH. In addition to showing that hypoxia can trigger neuroimmune mechanisms that possibly involve the microglia activity and also recruit the C1/PVH neural pathway.