The role of the hypothalamic paraventricular nucleus (PVN) in cardiovascular and neuroendocrine regulation has been well documented. Much remains unknown however about the integration of synaptic signals within this nucleus and the neuronal subtypes and chemical messengers governing these processes. Angiotensin II (ANG) has been demonstrated to act as a neurotransmitter in PVN where it exerts considerable influence on neuronal excitability. The studies within this thesis were undertaken to delineate the actions of ANG on the membrane properties of PVN neurons and its effect on synaptic transmission within this nucleus. We report that ANG activates a nitric oxide mediated negative feedback loop in the PVN. The magnitude of the depolarizing response to ANG appears to be dependant on this GABAergic inhibitory input demonstrating there exists within PVN an intrinsic negative feedback loop which modulates neuronal excitability in response to peptidergic excitation. We also demonstrate that the depolarizing response to ANG in magnocellular neurons is in part dependent upon increases in glutamatergic excitatory synaptic input. These data in combination highlight the multiple levels of synaptic integration controlling the output of magnocellular neurons in PVN. PVN also contains significant populations of neurosecretory parvocellular neurons which exercise considerable influence over the adenohypophysis and therefore neuroendocrine regulation. ANG caused an AT1 receptor mediated depolarization of these neurosecretory neurons. Voltage-clamp analysis revealed that ANG activated a non-selective cationic current and reduced a sustained potassium current characteristic of IK. These studies identify multiple post-synaptic modulatory sites through which ANG can influence the excitability of neurosecretory parvocellular PVN neurons. The findings in this thesis provide the framework for a cellular model of action of ANG within PVN to regulate the activity of this nucleus not only through direct cellular mediated ion channel interactions but also through modulation of synaptic input within the magnocellular system of PVN. / Thesis (Ph.D, Physiology) -- Queen's University, 2008-01-18 14:10:22.319

Previously, we demonstrated that exposure to morphine during gestation increases hypothalamic norepinephrine (NE) content and turnover rate in adult male rats and decreases these measures in adult females. To investigate the basis of these alterations, the present study examined the effects of prenatal exposure to morphine on tyrosine hydroxylase immunoreactivity (TH-IR) in the brains of adult male and female progeny. In male rats, prenatal morphine exposure significantly increased the density of TH-IR in cells and fibers in the caudal paraventricular nucleus of the hypothalamus (PVN) and locus coeruleus (LC), but had no effects in the lateral hypothalamus (LH). In female rats that were ovariectomized (OVX), prenatal morphine exposure significantly decreased the density of TH-IR in cells and fibers in the LC. Interestingly, an injection of estrogen in OVX control females reduced the mean optical density of TH-IR in the LC, but it was ineffective in drug-exposed females in the same brain region. Estrogen injections also reduced the mean optical density of TH-IR in the LH but not in the PVN of females, regardless of prenatal drug exposure. Thus, the present study suggests that prenatal morphine exposure induces long-term, sex-specific alterations in TH-IR in the PVN and LC of adult progeny.

The developing brain is highly sensitive to environmental influences. Unfavorable nutrition is one kind of stress that can cause acute metabolic disorders during the neonatal period [1,2,3] and severe diseases in later life [4,5]. These early life experiences occurring during heightened periods of brain plasticity help determine the lifelong structural and functional aspects of brain and behavior. In humans, for example, weight gain during the first week of life increased the propensity for developing obesity several decades later [5]. This susceptibility is, if not all, related to the dynamic reversible epigenetic imprints left on the histones [6,7,8], especially during the prenatal and postpartum period [9]. Histones are highly dynamic and responsive towards environmental stress [10,11]. Through covalent modification of the histone tail, histones are able to direct DNA scaffolding and regulate gene expression [10,12]. Thus far, various types of post translational modifications have been identified on various histones tails [12]. Among them, the methylation and acetylation on lysine residue (K) 27 on histone 3 (H3) has been tightly linked to gene repression [13,14] and activation [15], respectively. EZh2 (enhancer of zeste 2) in the polycomb repressive complex 2 (PRC2) is the only methyltransferase that has been linked to catalyze this methylation reaction. In addition, SUZ (suppressor of zeste) and EED (embryonic ectoderm development) are two other key proteins in PRC2 function core that help EZH2. As previous reported, increased H3K27 methylation was monitored after fasting stress during neonatal period in chicks' paraventricular nucleus (PVN). In this study, we investigated the detailed mechanism behind changes in H3K27 methylation following fasting stress. After 24 hours fasting on 3 days-of-age (D3), chicks exhibited elevated mRNA levels of PRC2 key components, including EZH2, SUZ and EED, in the PVN on D4. Western blots confirmed this finding by showing increased global methylation status at the H3K27 site in the PVN on D4. In addition, until 38 days post fasting, SUZ and EZH2 remained inhibited. A newly identified anorexigenic factor, Brain-derived neurotrophic factor (BDNF), was used as an example of multiple hormones expressed in PVN to verify this finding. Both BDNF protein and mRNA exhibited compatible changes to global changes of tri- (me3) and di-methylated (me2) H327. Furthermore, by using chromatin immunoprecipitation assays (ChIP), we were able to monitor the changes of H3K27me2/me3 deposition along the Bdnf gene. Fasting significantly increased H3K27me2/me3 as well as EZH2 at the Bdnf's promoter, transcription start site and 3'-untranslated region. These data show that fasting stress during the early life period could leave epigenetic imprinting in PVN for a long time. Next, we tried to understand the function of this epigenetic imprinting in the chicks' PVN. Thus, we compared naive chicks (never fasted) to chicks that received either a single 24 hour fast on D3 or two 24 hour fast on both D3 and 10 days-of-age (D10). We found that the D3 fasted group significantly increased the level of PRC2 key components and its product H3K27me2/me3 compared to the naive group. However, D3 fasting and D10 fasting together decreased the surges of H3K27me2/me3, SUZ and EED (not EZH2) compared to the naive group. We called this phenomenon "epigenetic memory". The Western blot, qPCR and CHIP assay results from BDNF all confirmed the existence of "epigenetic memory" for PRC2. These data suggested that fasting stress during the early period of brain development could leave long term epigenetic modifications in neurons. These changes could be beneficial to the body, which keeps homeostasis of inner environment and prevent massive response to future same stress. The EZH2 protein was knocked down and the H3K27 methylation status changes were monitored after applying the same treatment. We first confirmed that EZH2 antisense oligonucleotides (5.5 ug), but not EZH2 siRNA and artificial cerebrospinal fluid (ACSF), inhibit EZH2 protein by 86 % in the PVN. Then, on D3, chicks were subjected to a 24 hour fasting stress (D3-fasting) post either EZH2 antisense or ACSF injection. The EZH2 antisense blocked the surge of both EZH2 mRNA and H3K27 methylation after D3-fasting. At the same time, BDNF exhibited elevated expression levels and less methylated H3K27 deposition along the Bdnf gene. In addition, we were also interested in the changes of "epigenetic memory" post EZH2 antisense injection. We found that after EZH2 antisense injection, chicks' PVN no longer exhibited any "epigenetic memory" to repetitive fasting stress. While EZH2 mRNA was constantly inhibited, SUZ, EED and H3K27me2/3 levels were unpredictable. These findings suggested that neurons in the PVN utilized PRC2 as a major H3K27 methylation tool. Knockdown of EZH2 in the PRC2 impaired the proper response in PVN to fasting stress and PVN's ability to acclimate to repetitive fasting stresses. Thus, EZH2 is an important H3K27 methyltransferase inside chicken hypothalamus to maintain homeostasis. In conclusion, fasting stress during the early life period could leave epigenetic markers on chromosomes of neurons in the feeding regulation center. These epigenetic markers will be left on chromosomes for a long period of time and have a beneficial role in keeping homeostasis when individuals face future fasting stress again. H3K27 methylation is one of these epigenetic markers and inhibits expression of various genes inside neurons. EZH2 is so far the only detected methyltransferases for H3K27 that form the PRC2. Thus EZH2 plays a key function in the body's response to fasting. / Ph. D.

Abstract Statement of problem Plasma angiotensin II (Ang II) increases blood pressure (BP) through the activation of brain angiotensinergic pathways and the aldosterone-mineralocorticoid receptors (MR)- epithelial Na+ channel (ENaC)-endogenous ouabain (EO) pathway. The response of BP to circulating Ang II is enhanced by high salt intake, but the central mechanisms mediating this elevated response are not known. Methods of investigation Study 1) Male Wistar rats were divided into 4 groups and treated with regular salt diet (0.4% NaCl), high salt diet (2% NaCl), sc Ang II infusion (150 ng/kg/min), or sc Ang II infusion together with 2% salt diet for 14 days; plasma aldosterone and corticosterone levels, CYP11B2 mRNA in adrenal cortex and the mRNA levels of Ang II type 1 receptors (AT1R), CYP11B1 (11-β hydroxylase), CYP11B2 (aldosterone synthase), MR, 11βHSD2, ENaC α, ENaC β and ENaC γ in the subfornical organ (SFO), paraventricular nucleus (PVN), supraoptic nucleus (SON) and rostral ventrolateral medulla (RVLM) were measured. Study 2) MR blockers (eplerenone, spironolactone), ENaC blocker (benzamil), AT1R blocker (losartan) or vehicles were centrally infused in rats treated with Ang II plus high salt, and BP and heart rate (HR) were recorded by telemetry; plasma aldosterone and corticosterone levels and CYP11B2 mRNA expression in adrenal cortex were measured. Major findings Ang II alone caused a small increase in BP. Ang II together with 2% salt diet markedly increased the BP and plasma aldosterone level. Sc Ang II decreased 11βHSD2 and MR mRNA expression in the PVN, increased AT1R and ENaC γ expression in the PVN, and increased AT1R mRNA expression in the RVLM. Other genes tested in the four brain nuclei were not affected by sc Ang II or high salt diet. BP and plasma aldosterone increases in response to Ang II and salt, as well as CYP11B2 mRNA expression in adrenal cortex, were largely prevented by central infusion of eplerenone, spironolactone, benzamil or losartan. Main conclusion BP and plasma aldosterone responses to Ang II-salt are under the control of central mechanisms, and MR-AT1R activation in the brain plays a critical role in Ang II-salt induced hypertension.

No description available.

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)

Myocardial infarction (MI) induced heart failure (HF) is a leading cause of morbidity and mortality over the world. Regular exercise improves quality of life and decreases hospitalization and mortality of patients with HF. In animals, exercise post MI attenuates progressive cardiac remodeling and cardiac dysfunction, and decreases neuronal activity in the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM), which are key brain nuclei contributing to sympathetic hyperactivity post MI. The peripheral and central molecular mechanisms underlying these beneficial effects of exercise are not well understood. We studied one possible mechanism, brain-derived neurotrophic factor (BDNF), an exercise-induced factor, which via binding to its receptor tropomyosin-related kinase B (TrkB) may contribute to improvement of cardiac function post MI. In the brain, the ratio between two isoforms of the TrkB receptor, full-length and truncated forms (TrkB.FL/TrkB.T1) determines the extent of intracellular responses to mature BDNF (mBDNF; an active form of BDNF) and a decrease in this ratio may reflect down-regulation of BDNF-TrkB.FL signaling. Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase B (Akt) are intracellular factors of BDNF-TrkB signaling in hippocampal/cortical neurons. Activation of cardiac BDNF-TrkB signaling may increase cardiomyocyte survival and myocardial contractility. In hypertensive rats, the role of BDNF-TrkB signaling in the PVN and RVLM appears opposite with activation of this axis in the PVN increasing, but in the RVLM decreasing sympathetic nerve activity (SNA). However, activation of CaMKII and Akt in the PVN and RVLM both mediate increase in SNA. The specific role of BDNF-TrkB signaling in the PVN and RVLM of rats with HF post MI has not yet been studied. We hypothesized that exercise training post MI enhances BDNF-TrkB signaling pathways in the left ventricle (LV) and RVLM, but inhibits in the PVN, and thereby preserves cardiac structure and function post MI. We evaluated changes in BDNF-TrkB axis and intracellular factors CaMKII and Akt in the non-infarct area of the LV, PVN and RVLM in sedentary and exercising rats with MI. The impact of systemic blockade of BDNF-TrkB signaling was assessed with ANA-12, a selective non-competitive antagonist of TrkB receptors. In the infarct area of the LV, mBDNF protein decreased and TrkB.T1 protein increased. In the non-infarct area, mBDNF tended to be decreased without change in TrkB.T1 expression. The activities of CaMKII and Akt were decreased in the non-infarct area of the LV. In the PVN and RVLM, the TrkB.FL/TrkB.T1 ratio was decreased but without changes in mBDNF and downstream factors except for decrease in Akt activity in the RVLM. Exercise training improved ejection fraction (EF), cardiac index and LV end-diastolic pressure, but only the exercise-induced improvement of EF was blocked by ANA-12. In the non-infarct area of the LV, exercise prevented decreases in mBDNF, CaMKII and Akt, and these effects were prevented by ANA-12. In the PVN, exercise increased mBDNF and decreased Akt activity, whereas in the RVLM, exercise had no effect on mBDNF but decreased CaMKII activity. The exercise-induced increase mBDNF in the PVN and decrease in p-CaMKIIβ expression in the RVLM were prevented by ANA-12. Our findings suggest that down-regulation of BDNF-TrkB signaling post MI is prominent in the LV with decreases in mBDNF protein in the infarct area and intracellular factors CaMKII and Akt in the non-infarct area. Increases in mBDNF, CaMKII and Akt in the LV by exercise may contribute to improvement of EF. In the PVN and RVLM, despite a decrease in the ratio of TrkB.FL/TrkB.T1 in both brain nuclei, only Akt activity decreased in the RVLM post MI. Exercise-induced decreases in activities of CaMKII in the RVLM and Akt in the PVN may both contribute to reduction in sympathetic hyperactivity post MI.

A atrite reumatóide é uma doença inflamatória crônica, sem cura, que afeta cerca de 1% da população mundial entre 35 e 65 anos, cujos sinais e sintomas incluem dor, edema, rigidez, degeneração e deformidades articulares. O atual tratamento da artrite reumatóide consiste no uso de drogas anti-reumáticas modificadores de doença (DMARDs), porém são compostos caros e imunossupressivos que podem elevar o risco de infecções graves e malignidades. No presente estudo, analisamos o nervo vago como potencial imunomodulador da inflamação que ocorre na artrite reumatóide experimental. Nossos resultados indicam que a estimulação vagal aferente controla a inflamação articular por meio da ativação de áreas encefálicas simpatoexcitatórias, tais como o núcleo paraventricular do hipotálamo (PVN) e o locus coeruleus (LC). A estimulação do PVN ou do LC diminui a inflamação na articulação, mas somente a integridade do LC foi obrigatória para o controle vagal da inflamação na artrite. A estimulação elétrica cortical direcionada para o córtex parietal ativou o LC e o PVN, mimetizando a ativação vagal, porém induziu um melhor controle da inflamação. Esses resultados sugerem a existência de um mapa encefálico neuroimune capaz de controlar a artrite sem causar efeitos colaterais observáveis. / There is no cure for rheumatoid arthritis affecting over 1% of the world population between 35 e 65 years old suffering chronic inflammation causing pain, swelling, stiffness, degeneration e joint deformities. Disease-modifying anti-rheumatic drugs (DMARDs) are expensive e immunosuppressive, increasing the risk of severe infections e malignancies. Here, we analyzed the potential of the vagus nerve to control experimental arthritic inflammation. Our results indicate that the afferent vagus nerve controls arthritic joint inflammation by activating specific sympatho-excitatory brain areas, such as the paraventricular hypothalamic nucleus (PVN) e the locus coeruleus (LC). PVN or LC stimulation decreased articular inflammation, but only LC integrity was necessary for vagal control of arthritic inflammation. Cortical electrical stimulation above the parietal cortex activated LC e PVN, mimicked vagal activation but induced a better control of arthritic joint inflammation. These results suggest a neuroimmune brain map to control side-specific lateral arthritic joint inflammation without noticeable side effects.

A atrite reumatóide é uma doença inflamatória crônica, sem cura, que afeta cerca de 1% da população mundial entre 35 e 65 anos, cujos sinais e sintomas incluem dor, edema, rigidez, degeneração e deformidades articulares. O atual tratamento da artrite reumatóide consiste no uso de drogas anti-reumáticas modificadores de doença (DMARDs), porém são compostos caros e imunossupressivos que podem elevar o risco de infecções graves e malignidades. No presente estudo, analisamos o nervo vago como potencial imunomodulador da inflamação que ocorre na artrite reumatóide experimental. Nossos resultados indicam que a estimulação vagal aferente controla a inflamação articular por meio da ativação de áreas encefálicas simpatoexcitatórias, tais como o núcleo paraventricular do hipotálamo (PVN) e o locus coeruleus (LC). A estimulação do PVN ou do LC diminui a inflamação na articulação, mas somente a integridade do LC foi obrigatória para o controle vagal da inflamação na artrite. A estimulação elétrica cortical direcionada para o córtex parietal ativou o LC e o PVN, mimetizando a ativação vagal, porém induziu um melhor controle da inflamação. Esses resultados sugerem a existência de um mapa encefálico neuroimune capaz de controlar a artrite sem causar efeitos colaterais observáveis. / There is no cure for rheumatoid arthritis affecting over 1% of the world population between 35 e 65 years old suffering chronic inflammation causing pain, swelling, stiffness, degeneration e joint deformities. Disease-modifying anti-rheumatic drugs (DMARDs) are expensive e immunosuppressive, increasing the risk of severe infections e malignancies. Here, we analyzed the potential of the vagus nerve to control experimental arthritic inflammation. Our results indicate that the afferent vagus nerve controls arthritic joint inflammation by activating specific sympatho-excitatory brain areas, such as the paraventricular hypothalamic nucleus (PVN) e the locus coeruleus (LC). PVN or LC stimulation decreased articular inflammation, but only LC integrity was necessary for vagal control of arthritic inflammation. Cortical electrical stimulation above the parietal cortex activated LC e PVN, mimicked vagal activation but induced a better control of arthritic joint inflammation. These results suggest a neuroimmune brain map to control side-specific lateral arthritic joint inflammation without noticeable side effects.