O ATP mediando a sinalização purinérgica no bulbo ventrolateral rostral contribui para o controle do quimiorreflexo central e periférico regulando a pressão arterial e a respiração, mediante o envolvimento dos neurônios do núcleo retrotrapezóide (RTN). No entanto, as potenciais contribuições da sinalização purinérgica, no RTN, na função cardiorrespiratória em animais não anestesiados ainda não foram testadas. Mostramos que a injeção de ATP no RTN promoveu aumento cardiorrespiratório por um mecanismo dependente de receptores P2. Mostramos também que o bloqueio de receptor P2 não específico (PPADS), mas não de receptores específicos P2Y (MRS2179), reduziu a resposta ventilatória à hipercapnia (7% CO2) e hipóxia (8 % O2) em ratos não anestesiados. Além disso, a adenosina (ADO) no RTN atenuou o aumento da ventilação induzido por hipercapnia in vivo e o disparo dos neurônios in vitro. Estes resultados demonstram que a sinalização mediada por ATP contribui para o controle respiratório do quimiorreflexo central e periférico em ratos acordados e uma vez que o ATP se metaboliza rapidamente em ADO, esta teria ação no balanço da resposta quimiorreceptora no RTN. / ATP-mediated purinergic signaling at the level of the rostral ventrolateral medulla (RVLM) contributes to both central and peripheral chemoreceptor control of breathing and blood pressure within the retrotrapezoid nucleus (RTN). However, potential contributions of purinergic signaling in the RTN to cardiorespiratory function in conscious animals has not been tested. We show that in the absence of functional C1 cells, ATP into the RTN increased cardiorespiratory output by a P2-recepor dependent mechanism. We also show that a non-specific P2 receptor blocker (PPADS) reduced the ventilatory response to hypercapnia (7% CO2) and hypoxia (8% O2) in unanesthetized awake rats. Conversely, a specific P2Y1-receptor blocker (MRS2179) into the RTN had no measurable effect on respiratory responses elicited by hypercapnia or hypoxia. Moreover, adenosine (ADO) into the RTN could attenuate the hypercapnia-induced increase in ventilation in vivo and firing rate in RTN neurons in vitro. These results demonstrate that ATP-mediated purinergic signaling contributes to central and peripheral chemoreflex control of breathing in awake rats and ADO could provide a balance between ATP stimulation and its inhibition in RTN during hipercapnia.

O núcleo retrotrapezóide (RTN) é uma região bulbar envolvida na respiração. Estudos prévios mostraram a presença de varicosidades catecolaminérgicas na região do RTN. O objetivo deste estudo foi investigar a fonte de catecolaminas e os efeitos promovidos pela ativação dos receptores adrenérgicos no RTN. Uma densa projeção neuronal do grupamento A7 para o RTN foi revelada usando o traçador retrógrado Fluorogold. Foi registrada a atividade eletromiográfica do diafragma (DiaEMG) e do abdominal (AbdEMG) de ratos Wistar anestesiados. A injeção de noradrenalina promoveu uma inibição da DiaEMG, sem alterar a AbdEMG; este efeito foi atenuado pela injeção prévia de ioimbina e não foi afetado pela injeção de prazosina e propranolol no RTN. A injeção de fenilefrina no RTN aumentou a DiaEMG e gerou AbdEMG; estes efeitos foram bloqueados por injeções prévias de prazosina no RTN. Os resultados deste estudo suportam a ideia de que o RTN recebe projeções adrenérgicas da ponte que modula a atividade dos neurônios do RTN por meio da ativação dos receptores adrenérgicos α -1 e α- 2. / The retrotrapezoid nucleus (RTN) is a medulla region involved in breathing. Previous studies showed the presence of catecholaminergic varicosities in the RTN region. The aim of this study was to investigate the source of cathecolamines and the effects produced by the activation of adrenergic receptors in the RTN. A dense neuronal projection from A7 to RTN was revealed using retrograde tracer FluorGold. In anaesthetized male Wistar rats, diaphragm (DiaEMG) and abdominal (AbdEMG) muscle activities were recorded. Injection of noradrenaline produced an inhibition of DiaEMG, but did not change AbdEMG; These effects was attenuated by pre-injection of yohimbine and were not affect by injection of prazosin and propranolol into the RTN. Injection of phenilephrine into the RTN increased DiaEMG and was also able to generate AbdEMG; these responses were eliminated by pre-injections of into the RTN. These results support the idea that RTN has pontine adrenergic inputs that modulate RTN neurons activity through activation of &#945 - 1 and - &#945 -2 adrenergic receptors.

by Perpetua E. Tan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 149-157). / Abstract --- p.9 / Acknowledgments --- p.12 / List of abbreviations --- p.13 / List of Tables --- p.16 / List of Figures --- p.19 / Chapter CHAPTER 1 --- INTRODUCTION --- p.21 / Chapter CHAPTER 2 --- LITERATURE REVIEWS CATECHOLAMINES: NORADRENALINE AND ADRENALINE --- p.25 / Chapter 2.1 --- History --- p.25 / Chapter 2.2 --- Origin of plasma catecholamines --- p.25 / Chapter 2.3 --- Kinetics of entry and removal --- p.28 / Chapter 2.4 --- Levels present in plasma --- p.30 / Chapter 2.5 --- Some factors affecting plasma CA levels --- p.31 / Chapter 2.5.1 --- Effects of age --- p.31 / Chapter 2.5.2 --- Postural change --- p.32 / Chapter 2.5.3 --- Exercise --- p.32 / Chapter 2.5.4 --- Temperature change --- p.32 / Chapter 2.5.5 --- Stress --- p.33 / Chapter 2.5.6 --- Pregnancy --- p.34 / Chapter 2.5.7 --- Disease --- p.35 / Chapter 2.6 --- Actions in the body --- p.35 / Chapter 2.6.1 --- Plasma endogenous catecholamines --- p.35 / Chapter 2.6.2 --- Plasma exogenous catecholamines and medicine --- p.36 / Chapter 2.6.2.1 --- Clinical uses --- p.36 / Chapter 2.6.2.2 --- Effects --- p.37 / Chapter 2.6.2.3 --- Side effects --- p.38 / Chapter 2.7 --- Binding of catecholamines in plasma --- p.38 / Chapter 2.8 --- Measurement of catecholamines in plasma --- p.38 / Chapter 2.8.1 --- Chemistry --- p.38 / Chapter 2.8.2 --- Extraction and purification --- p.39 / Chapter 2.8.3 --- Biological methods --- p.40 / Chapter 2.8.4 --- Colorimetry --- p.41 / Chapter 2.8.5 --- Radioimmunoassay and radioenzymatic assay --- p.41 / Chapter 2.8.6 --- Enzyme-linked immunoassay --- p.42 / Chapter 2.8.7 --- Gas chromatography --- p.42 / Chapter 2.8.8 --- Liquid chromatography --- p.42 / Chapter 2.8.8.1 --- Fluorometry --- p.43 / Chapter 2.8.8.2 --- Electrochemical detection --- p.43 / Chapter 2.9 --- Plasma protein binding of basic drugs --- p.44 / Chapter 2.9.1 --- Binding to albumin --- p.45 / Chapter 2.9.2 --- Binding to alpha-1-acid-glycoprotein --- p.45 / Chapter 2.9.3 --- Binding to other proteins --- p.45 / Chapter 2.9.4 --- Factors affecting drug binding --- p.46 / Chapter 2.9.4.1 --- Pregnancy --- p.46 / Chapter 2.9.4.2 --- Age --- p.46 / Chapter 2.9.4.3 --- Disease states --- p.46 / Chapter 2.9.5 --- Separation procedures to reveal and follow drug protein binding --- p.47 / Chapter 2.9.5.1 --- Equilibrium dialysis --- p.47 / Chapter 2.9.5.2 --- Ultrafiltration --- p.48 / Chapter 2.9.5.3 --- Ultracentrifugation --- p.48 / Chapter 2.9.5.4 --- Gel Filtration --- p.48 / Chapter CHAPTER 3 --- ANALYTICAL TECHNIQUE : PLASMA CATECHOLAMINE ANALYSIS --- p.49 / Chapter 3.1 --- HPLC determination with coulometric detection of catecholamines --- p.49 / Chapter 3.1.1 --- Introduction --- p.49 / Chapter 3.1.2 --- Basic equipment --- p.49 / Chapter 3.1.3 --- Mobile phase preparation --- p.50 / Chapter 3.1.3.1 --- Reagent A (Citrate-acetate-EDTA buffer) --- p.50 / Chapter 3.1.3.2 --- Reagent B (ion pairing reagent) --- p.50 / Chapter 3.1.3.3 --- Mobile phase mixture --- p.50 / Chapter 3.1.4 --- Detector settings --- p.51 / Chapter 3.1.5 --- Sample collection and storage --- p.51 / Chapter 3.2 --- Reagents and solutions --- p.52 / Chapter 3.2.1 --- Acid-washed alumina --- p.52 / Chapter 3.2.2 --- Tris buffer solution --- p.53 / Chapter 3.2.3 --- Washing solution --- p.53 / Chapter 3.2.4 --- Acetic acid solution --- p.53 / Chapter 3.2.5 --- EDTA-HC1 solution --- p.53 / Chapter 3.2.6 --- Citric acid solution --- p.53 / Chapter 3.2.7 --- Stock solutions --- p.54 / Chapter 3.2.7.1 --- Catecholamine standards --- p.54 / Chapter 3.2.7.2 --- Dihydroxybenzylamine (Internal) standard --- p.54 / Chapter 3.2.8 --- Stripped fresh frozen plasma --- p.54 / Chapter 3.2.9 --- Sorensen's phosphate buffer containing 0.6% NaCl --- p.55 / Chapter 3.2.10 --- Control standards --- p.55 / Chapter 3.3 --- Voltammogram of catecholamines and internal standard used --- p.55 / Chapter 3.4 --- Maintenance of the HPLC-Coulometric detector system --- p.56 / Chapter 3.5 --- Optimization of the extraction method --- p.58 / Chapter 3.5.1 --- Amount of alumina for adsorption of CA --- p.58 / Chapter 3.5.2 --- pH of tris buffer for maximum uptake of CA onto alumina --- p.58 / Chapter 3.5.3 --- Optimum time for maximum uptake of CA onto alumina --- p.59 / Chapter 3.5.4 --- Optimum time for maximum desorption of CA into acid solution --- p.59 / Chapter 3.5.5 --- Optimum volume of acid solution for maximum desorption of CA --- p.60 / Chapter 3.6 --- Validation of the method --- p.60 / Chapter 3.6.1 --- Linearity --- p.60 / Chapter 3.6.2 --- Recovery --- p.61 / Chapter 3.6.3 --- Reproducibility --- p.62 / Chapter 3.6.4 --- Stability --- p.62 / Chapter 3.7 --- Results --- p.63 / Chapter 3.8 --- Discussion --- p.79 / Chapter CHAPTER 4 --- CLINICAL APPLICATIONS OF THE CATECHOLAMINE ASSAY --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.1.1 --- Applications of catecholamines assay in clinical science --- p.84 / Chapter 4.2 --- : PLASMA CATECHOLAMINES AFTER INDUCTION OF ANAESTHESIA AT CAESARIAN SECTION --- p.84 / Chapter 4.2.1 --- Introduction --- p.84 / Chapter 4.2.2 --- Patients and methods --- p.86 / Chapter 4.2.3 --- Blood sampling and storage --- p.87 / Chapter 4.2.4 --- Statistics used --- p.87 / Chapter 4.2.5 --- Results --- p.88 / Chapter 4.2.6 --- Discussion --- p.99 / Chapter 4.3 --- EPINEPHRINE INFILTRATION IN SINUS SURGERY --- p.101 / Chapter 4.3.1 --- Introduction --- p.101 / Chapter 4.3.2 --- Patients and methods --- p.102 / Chapter 4.3.3 --- Blood sampling and storage --- p.103 / Chapter 4.3.4 --- Results --- p.104 / Chapter 4.3.5 --- Discussion --- p.108 / Chapter CHAPTER 5 --- ANALYTICAL TECHNIQUE: PLASMA PROTEIN BINDING OF CATECHOLAMINES --- p.110 / Chapter 5.1 --- Equilibrium dialysis for protein binding of drugs --- p.110 / Chapter 5.1.1 --- Introduction --- p.110 / Chapter 5.1.2 --- Dialyzing apparatus --- p.110 / Chapter 5.1.3 --- Sample collection and storage --- p.111 / Chapter 5.1.4 --- Reagents and solutions --- p.111 / Chapter 5.1.4.1 --- Ascorbic acid --- p.111 / Chapter 5.1.4.2 --- Glutathione --- p.111 / Chapter 5.1.4.3 --- Sodium metabisulfite --- p.111 / Chapter 5.1.4.4 --- Dialysis buffer --- p.111 / Chapter 5.1.5 --- Dialysis membrane --- p.112 / Chapter 5.1.6 --- Equilibrium dialysis --- p.112 / Chapter 5.2 --- Optimization of the binding parameters --- p.113 / Chapter 5.2.1 --- Types of preservatives for stability of catecholamines during dialysis --- p.113 / Chapter 5.2.2 --- Dialysis buffer --- p.114 / Chapter 5.2.3 --- Dialysis time and volume of sample --- p.114 / Chapter 5.2.4 --- Dialysis membrane --- p.115 / Chapter 5.2.5 --- Catecholamines concentration for dialysis --- p.114 / Chapter 5.3 --- Total protein analysis- Lowry Method --- p.115 / Chapter 5.3.1 --- Reagents and solutions --- p.116 / Chapter 5.3.1.1 --- Reagent A (Alkaline copper reagent) --- p.116 / Chapter 5.3.1.2 --- Reagent B (Folin-Ciocalteus phenol reagent with water) --- p.116 / Chapter 5.3.2 --- Stock standard and controls --- p.116 / Chapter 5.3.2.1 --- Human serum albumin standard --- p.116 / Chapter 5.3.2.2 --- Controls --- p.116 / Chapter 5.3.3 --- Procedure --- p.116 / Chapter 5.4 --- Results --- p.117 / Chapter 5.5 --- Discussion --- p.126 / Chapter CHAPTER 6 --- CONCLUSIONS --- p.130 / APPENDIX --- p.134 / CHEMICALS AND REAGENTS --- p.146 / REFERENCES --- p.149

by Cheung Ngai Man, Edmund. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 73-83). / Acknowledgments --- p.i / Abstract --- p.iii / List of Figures --- p.viii / List of Tables --- p.xi / Chapters / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Intramedullary nailing --- p.1 / Chapter 1.2 --- Reaming technique for intramedullary nailing --- p.3 / Chapter 1.3 --- The relationship between pulmonary fat embolism and reaming technique --- p.7 / Chapter 1.4 --- Objectives --- p.10 / Chapter 2 --- Methodology --- p.12 / Chapter 2.1 --- The measurement of the intramedullary pressure --- p.12 / Chapter 2.1.1 --- Animal model --- p.12 / Chapter 2.1.2 --- Intramedullary pressure measurement device --- p.12 / Chapter 2.1.3 --- Operative procedure --- p.14 / Chapter 2.1.4 --- Intramedullary pressure measurement --- p.16 / Chapter 2.2 --- The measurement of the plasma lipids and marrow lipids --- p.19 / Chapter 2.2.1 --- Samples collection --- p.19 / Chapter 2.2.2 --- Lipid extraction --- p.19 / Chapter 2.2.3 --- Thin layer chromatography --- p.20 / Chapter 2.2.4 --- Methylation --- p.24 / Chapter 2.2.5 --- Gas chromatographic analysis --- p.24 / Chapter 2.3 --- The measurement of the pulmonary lipids and fat emboli --- p.27 / Chapter 2.3.1 --- Pulmonary tissue collection --- p.27 / Chapter 2.3.2 --- Preparation for measurement of pulmonary lipids --- p.27 / Chapter 2.3.3 --- Fat emboli staining --- p.27 / Chapter 2.3.4 --- Image analysis --- p.28 / Chapter 2.4 --- Statistical analysis --- p.31 / Chapter 3 --- Results --- p.32 / Chapter 3.1 --- Intramedullary pressure measurement --- p.32 / Chapter 3.2 --- The analysis of bone marrow lipids --- p.34 / Chapter 3.3 --- The changes of the plasma lipids during reaming --- p.39 / Chapter 3.4 --- The measurement of the pulmonary fat emboli --- p.44 / Chapter 3.5 --- The relationship between the intramedullary pressure and plasma lipids and pulmonary fat intravasation --- p.52 / Chapter 4 --- Discuss --- p.55 / Chapter 4.1 --- The experimental design --- p.55 / Chapter 4.2 --- The change of the intramedullary pressures --- p.57 / Chapter 4.3 --- The application of the gas chromatography --- p.59 / Chapter 4.4 --- The composition of bone marrow lipids --- p.62 / Chapter 4.5 --- The changes of plasma lipids --- p.63 / Chapter 4.6 --- The pulmonary fat embolisation --- p.65 / Chapter 5 --- Conclusion --- p.69 / Chapter 6 --- Future direction on this study --- p.71 / References --- p.73 / Appendix --- p.84 / Chapter 1 --- The operation of the IM Press device --- p.84 / Chapter 2 --- The calibration of the IM Press --- p.85 / Chapter 3 --- The preparation of the internal standards for the lipid analysis --- p.89 / Chapter 4 --- The composition of the bone marrow lipids --- p.91 / Chapter 5 --- The composition of plasma lipids --- p.95 / Chapter 6 --- The composition of pulmonary lipids --- p.101 / Chapter 7 --- The measurement of the pulmonary fat emboli --- p.105

O núcleo retrotrapezóide (RTN) é uma região bulbar envolvida na respiração. Estudos prévios mostraram a presença de varicosidades catecolaminérgicas na região do RTN. O objetivo deste estudo foi investigar a fonte de catecolaminas e os efeitos promovidos pela ativação dos receptores adrenérgicos no RTN. Uma densa projeção neuronal do grupamento A7 para o RTN foi revelada usando o traçador retrógrado Fluorogold. Foi registrada a atividade eletromiográfica do diafragma (DiaEMG) e do abdominal (AbdEMG) de ratos Wistar anestesiados. A injeção de noradrenalina promoveu uma inibição da DiaEMG, sem alterar a AbdEMG; este efeito foi atenuado pela injeção prévia de ioimbina e não foi afetado pela injeção de prazosina e propranolol no RTN. A injeção de fenilefrina no RTN aumentou a DiaEMG e gerou AbdEMG; estes efeitos foram bloqueados por injeções prévias de prazosina no RTN. Os resultados deste estudo suportam a ideia de que o RTN recebe projeções adrenérgicas da ponte que modula a atividade dos neurônios do RTN por meio da ativação dos receptores adrenérgicos α -1 e α- 2. / The retrotrapezoid nucleus (RTN) is a medulla region involved in breathing. Previous studies showed the presence of catecholaminergic varicosities in the RTN region. The aim of this study was to investigate the source of cathecolamines and the effects produced by the activation of adrenergic receptors in the RTN. A dense neuronal projection from A7 to RTN was revealed using retrograde tracer FluorGold. In anaesthetized male Wistar rats, diaphragm (DiaEMG) and abdominal (AbdEMG) muscle activities were recorded. Injection of noradrenaline produced an inhibition of DiaEMG, but did not change AbdEMG; These effects was attenuated by pre-injection of yohimbine and were not affect by injection of prazosin and propranolol into the RTN. Injection of phenilephrine into the RTN increased DiaEMG and was also able to generate AbdEMG; these responses were eliminated by pre-injections of into the RTN. These results support the idea that RTN has pontine adrenergic inputs that modulate RTN neurons activity through activation of &#945 - 1 and - &#945 -2 adrenergic receptors.

Chronic pain is a major healthcare concern that directly affects over one hundred million people in the United States alone. While current treatment options like opioids and NSAIDs are effective, they are with significant drawbacks that prevent long term use. It is important to identify and understand new druggable targets for the treatment of pain. Recent findings have demonstrated substance P functions in the RVM to maintain hypersensitivity to noxious heat stimuli in models of persistent peripheral inflammatory injury in a manner dependent on presynaptic NMDA receptors. What remains unclear is how substance P assumes this pronociceptive role following peripheral inflammatory injury. The experiments detailed in this thesis investigated whether the levels and or release of substance P in the RVM was altered following peripheral inflammatory injury. The effect of peripheral inflammatory injury on levels of substance P in the RVM was tested at several time points. The data show that there were no changes in substance P levels in the ipsilateral or contralateral RVM of CFA injected rats compared to their saline controls at any of the time points tested. To assess whether changes in substance P levels occurred in a subset of neurons within the RVM, computer aided densitometry analysis was used to measure substance P immunoreactivity in sections from the RVM of rats treated with CFA or saline. Substance P immunoreactivity was increased in the ipsilateral RVM of the CFA group compared to the corresponding saline sections at the 4 day, but not the 2 week time point. No other changes were observed. Electron microscopy was used to demonstrate the presence of the NMDA receptor and substance P on the same axon terminals within the RVMs of rats treated with either CFA or saline. This colocalization is significant because it identifies NMDA receptors in position to regulate the release of substance P from axon terminals in the RVM. There were no obvious differences in the degree of colocalization between CFA and saline groups. Functional experiments were devised that tested whether substance P release (basal and evoked) in the RVM was increased following peripheral inflammatory injury, and whether said release was regulated by NMDA receptors. The data show that neither basal nor evoked (potassium or veratridine) release was increased following peripheral inflammatory injury. NMDA was able to facilitate the release of substance P in both the CFA and saline treatment groups, but the facilitation was not different between groups. In the absence of any depolarization stimulus, NMDA was unable to elicit any release of substance P beyond basal values. All told, the data show substance P levels in the RVM are not altered by peripheral inflammatory injury. Additionally, neither basal nor evoked release of substance P is altered by peripheral inflammatory injury. The data provide functional and anatomical evidence for modulation of substance P release by glutamate acting at presynaptic NMDA receptors, but do not support the idea of differential modulation of substance P release following peripheral inflammatory injury.

Doctor of Philosophy(PhD) / This thesis addresses the neurochemistry and function of specific nuclei in the autonomic nervous system that are crucial mediators of cardiorespiratory regulation. The primary aim is to build on previous knowledge about muscarinic cholinergic mechanisms within cardiorespiratory nuclei located in the ventrolateral medulla oblongata. The general focus is characterisation of gene expression patterns of specific muscarinic receptor subtypes in central nuclei involved in blood pressure control and respiratory control in normal rats. The findings were subsequently extended by characterisation of muscarinic receptor gene expression patterns in 1) a rat model of abnormal blood pressure control (hypertension) (Chapter 3) 2) a rat model of cholinergic sensitivity (Chapter 5) 3) the rat ventral respiratory group (Chapter 6) The results of a series of related investigations that ensued from the initial aims more finely characterise the neurocircuitry of the ventrolateral medulla, from a specifically cholinoceptive approach. All five muscarinic receptor subtypes are globally expressed in the ventrolateral medulla but only the M2R mRNA was significantly elevated in the VLM of hypertensive animals compared to their normotensive controls and in the VLM of animals displaying cholinergic hypersensitivity compared to their resistant controls. Surprisingly, M2R mRNA is absent in catecholaminergic cell groups but abundant in certain respiratory nuclei. Two smaller projects involving gene expression of other neurotransmitter / neuromodulators expressed in cardiorespiratory nuclei were also completed during my candidature. Firstly, the neurochemical characterisation of enkephalinergic neurons in the RVLM, and their relationship with bulbospinal, catecholaminergic neurons in hypertensive compared to normotensive animals was carried out (Chapter 4). A substantial proportion of sympathoexcitatory neurons located in the RVLM were enkephalinergic in nature. However, there was no significant difference in preproenkephalin expression in the RVLM in hypertensive compared to normotensive animals. Secondly, the identification and distribution of components of the renin-angiotensin aldosterone system (RAAS) within the brainstem, and differences in gene expression levels between hypertensive and normotensive animals was also investigated. The RAAS data was not included in this thesis, since the topic digresses substantially from other chapters and since it is published (Kumar et al., 2006). The mRNA expression aldosterone synthase, mineralocorticoid receptor (MR1), 12-lipoxygenase (12-LO), serum- and glucocorticoid- inducible kinase and K-ras) were found to be present at all rostrocaudal levels of the ventrolateral medulla. Expression of MR1 mRNA was lower in the RVLM of SHR compared with WKY rats and 12-LO mRNA levels were lower in the CVLM in SHR compared with WKY rats. Otherwise, there was no difference in gene expression level, or the method of detection was not sensitive enough to detect differences in low copy transcripts between hypertensive and normotensive animals.

The hypothalamic paraventricular nucleus (PVN) is known to be a major integrative region within the forebrain. It is composed of functionally different subgroups of neurons, including the parvocellular neurons that project to important autonomic targets in the brainstem e.g. the rostral ventrolateral medulla (RVLM) and the intermediolateral cell column (IML) of the spinal cord, where the sympathetic preganglionic motor-neurons are located. These regions are critical in cardiovascular regulation; hence, these projections are likely to mediate the effects of the PVN on sympathetic nerve activity and hence may contribute to the cardiovascular changes induced by physiological stimuli such as elevations in body temperature. The neurotransmitter such as nitric oxide (NO) is important in cardiovascular regulation and it is now emerging as a major focus of investigation in thermoregulation. One of the most striking accumulations of NO containing-neurons is in the PVN where it appears to be playing an important role in cardiovascular regulation and body fluid homeostasis. The results of the work show; 1. That spinally-projecting and nitrergic neurons in the PVN may contribute to the central pathways activated by exposure to a hot environment. 2. Suggests that nitrergic neurons and spinally- projecting neurons in the brainstem may make a small contribution to the central pathways mediating the reflex responses initiated by hyperthermia. 3. The present study also illustrates that these PVN neurons projecting to the RVLM may make a smaller contribution than the spinal-projecting neurons in the PVN to the cardiovascular responses initiated by heat. 4. The results of my studies showed that the microinjection of muscimol to inhibit the neuronal activity in the PVN abolished the reflex decrease in renal blood flow following an elevation of core body temperature. In addition, this effect was specific to the PVN, since microinjections of muscimol into areas outside the PVN were not effective. These findings demonstrate that the PVN is critical for this reflex cardiovascular response initiated by hyperthermia. In conclusion, PVN is critical for the reflex decrease in renal blood flow during elevations in core body temperature. We hypothesise that projections from the PVN to the spinal cord and the RVLM contribute to the reflex cardiovascular responses. Additionally, nitrergic neurons in the PVN may contribute but the physiological role of those neurons in the reflex responses elicited by hyperthermia needs to be investigated.

Morphine is the most widely prescribed opiate for alleviation of persistent pain; however, it is becoming increasingly clear that morphine is less potent in women compared to men. Morphine primarily binds mu opioid receptors, which are densely localized in the midbrain periaqueductal gray (PAG). Anatomical and physiological studies conducted in the 1960s identified the PAG, and its projections to the rostral ventromedial medulla (RVM) and spinal cord dorsal horn, as an essential neural circuit mediating opioid-based analgesia. Remarkably, the majority of studies since then were conducted in males with the implicit assumption that this circuit was the same in females; this is not the case. It is now well established that morphine produces greater analgesia in males compared to females in a wide range of vertebrates, however, the mechanism(s) driving this sex difference is not clear. Our recent studies indicate that two factors appear to be contributing to the sexually dimorphic effects of morphine. First, there are sex differences in the anatomy and physiology of the descending inhibitory pathway on which morphine acts to produce analgesia. Specifically, the projections from the PAG to the RVM are sexually dimorphic and activated to a greater degree by both inflammatory pain and systemic morphine in males. In the absence of pain, the PAG-RVM circuit is activated to a greater degree in males compared to females, while this activation steadily declines during the development of tolerance in males only. We also have evidence of a sexually dimorphic expression of mu opioid receptor within the PAG that appears to contribute to sex differences in morphine potency. Microinjection of morphine directly into the PAG produces significantly greater analgesia in males, indicating that the PAG is sufficient for eliciting this sexually dimorphic behavior. Furthermore, mu opioid receptor-expressing PAG neurons are necessary for eliciting a sexually dimorphic response to morphine as lesioning mu opioid receptor-expressing neurons attenuates analgesia in males only. Together, these data indicate that the PAG-RVM pathway and mu opioid receptor expression in the PAG is sexually dimorphic and provides a primary mechanism for sex differences in morphine potency.

An individual who has sustained either irreversible cessation of circulatory and respiratory functions, or irreversible cessation of all functions of the entire brain, including the brain stem is dead. Brain death is currently the legal definition of death in many countries. Many people confuse brain death with vegetative states. Patients in a vegetative state are unaware of themselves or their environment. Both patients with brain death and those in a vegetative state are unconscious following severe brain injury. Unlike the brain death, vegetative patient¡¦s vital vegetative functions, such as cardiac action, respiration, and maintenance of blood pressure are preserved. The rostral ventrolateral medulla (RVLM) is the origin of a ¡§life-and-death¡¨ signal identified from systemic arterial blood pressure spectrum and intimately related to brain death. Based on the animal models of brain death, the observations that the power density of the vasomotor components of SAP signals undergoes both augmentation and reduction during the progression towards death strongly suggest that both ¡¥¡¥pro-life¡¦¡¦ and ¡¥¡¥pro-death¡¦¡¦ programs are present in the RVLM. A number of those ¡¥¡¥pro-life¡¦¡¦ and ¡¥¡¥pro-death¡¦¡¦ programs in the RVLM has now been identified along with their cellular and molecular mechanisms. As the neural substrate that is intimately related to brain death, one unresolved question is whether the proteome expressed in RVLM is unique. To address the issue, we used the cerebral cortex, which is defunct under persistent vegetative state for comparison. 2-DE electrophoresis, MALTI-TOF MS and peptide mass fingerprinting were used for investigation the proteomic difference between the rat RVLM and cerebral cortex. Quantitative analysis on silver-stained 2-DE electrophoresis gels revealed highly comparable distribution patterns of these protein spots for both brain regions, with 85.9 ¡Ó 2.3 % of protein spots from RVLM matched those from cerebral cortex. According to the protein function, these proteins were classed into binding activity, chaperone, antioxidant, oxidoreductase, ubiquitin- proteasome system, cell cycle, catalytic activity, glycolysis, tricarboxylic acid cycle, electron transport chain, endocytosis and exocytosis, structural molecular function, apoptosis, transport, differentiation and neurogenesis, protein biosynthesis, cell junction, and others. We found that a group of antioxidant proteins, including members of the peroxiredoxin (Prx) family (Prx-1, Prx-2, Prx-5, and Prx-6), thioredoxin and mitochondrial manganese superoxide dismutase exhibited significantly higher protein and mRNA expression levels in RVLM when compared to cerebral cortex. Tissue oxygen, ATP contents and ATP synthase subunits alpha and beta in RVLM were also significantly elevated. On the other hand, protein and mRNA levels of members of the ubiquitin-proteasome system, including proteasome subunit alpha type-1, ubiquitin, uniquitin-conjugating enzyme E2 N, ubiquitin carboxyl-terminal hydrolase isozyme L1 and L3, were comparable in both brain regions. The presence of higher levels of tissue oxygen and ATP synthase subunits in RVLM, leading to augmented ATP production, provides a cellular safeguard mechanism to reduce the possibility of irreversible reduction in intracellular ATP contents that precipitate brain death. By manifesting an augmented tissue oxygen and metabolic energy production, RVLM is more prone to oxidative stress. We conclude that a significantly elevated level of antioxidant proteins and mRNA in RVLM is consistent with the exhibition of higher tissue oxygen tension and metabolic energy production in this neural substrate, which together constitute a safeguard mechanism against brain death.