Patrice G. Guyenet - US grants

The ability of the CNS to regulate peripheral haemodynamics is obviously insufficient to maintain arterial pressure within normal levels in hypertensive states. Moreover it is becoming increasingly evident that the CNS in fact contributes to the development and maintenance of the hypertension either because of genetic abnormalities (spontaneously hypertensive rats) or because it receives abnormal signals from the periphery (reset baroreceptors, excessive renal afferent inputs, excessive angiotensin levels etc.) which reflexly trigger inappropriate hormonal or sympathetic responses. The point of the present proposal is to focus on three regulatory mechanisms and to investigate their alterations in several hypertensive models in the rat. The first project consists in comparing the haemodynamic characteristics and sympathetic activity of two angiotensin II-dependent hypertensive models (2 KIC rats and rats rendered hypertensive via low dose infusion of angiotensin-II). Its main purpose will be to determine the specific contribution of A-II in renal hypertension. The second part consists in a neurophysiological investigation of the cardiovascular integration which takes place in the anterior ventrolateral medulla at the level of identified medullospinal sympathetic efferents. This study will be performed in SHR's, renal hypertensive animals (2 KIC rats) and animals rendered hypertensive by continuous infusions of A-II. Its object will be to determine directly the potential contribution of identified central sympatho-motor neurons to the hypertension. The third project consists in studying with molecular biological approaches the role of the central renin-angiotensin system. The presence of mRNA coding for angiotensin-II containing peptides and the exact nature of these molecules will be investigated. The regional distribution of this synthetic ability and its potential alteration in hypertension will be studied in several animal models including SHR's and AII-dependent hypertensive models (2 KIC rats and animals rendered hypertensive by infusion by low A-II doses). These three projects are presented as a single package because their completion requires a pool of techniques unavailable to any given contributing laboratory separately.

The long-term objective of this research is to understand the specific role played by catecholamines (CA) in information processing by the central nervous system. The proposed research will focus in large part on: i) the role of the CA projections to the intermediolateral cell column of the spinal cord (IML) (nucleus of orgin of the sympathetic preganglionic neurons, SPN's) and on ii) the mechanism of central Alpha-2 adrenergic postsynaptic inhibition (as it is presumably involved in the IML and possibly in the effects of the commonly used antihypertensive drugs clonidine and Alpha methyl DOPA). The nature and origin of these CA projections as well as the type of synaptic contacts established with the SPN's or the surrounding neuropil will be studied in the rat with neuroanatomical techniques combined with specific lesions. The effect of the various catecholamines on SPN's discharges will be studied with single-unit recordings and iontophoresis in the cat to examine the type of CA receptors involved in their action. In order to determine whether E or NE neurons are involved or not in the baroceptor reflex single unit recordings will be obtained from these neurons (C1 and A5) in the rat and the effect of pharmacologically induced alterations in blood pressure on their discharges will be investigated. For the same purpose an attempt will be made to determine the release of NE in the IML of the cat by voltammetry "in vivo" and its modification by changes in peripheral blood pressure. Finally a tissue slice preparation of the pontine area (species to be determined) will be set up to study with intracellular techniques the mechanism of Alpha-2 mediated postsynaptic inhibition in the locus coeruleus. These experiments will contribute to further our understanding of the control by the CNS of the sympathetic outflow and of the mechanism of action of Alpha-2 adrenergic agonists.

The role of the central nervous system in regulating peripheral haemodynamics has been studied extensively in normotensive and hypertensive subjects but this work has been mostly conducted from a global operational point of view (input-output relationships). Rather little is known about the specific CNS circuits involved. Such well known realities as the sympathetic tone, baroreceptor reflexes, central baroreceptor resetting (drug-induced or otherwise), the "vasomotor center", have as yet very imprecise neurophysiological substrates. Even the most basic medullary circuits, including the pathways projecting to the sympathetic preganglionic neurons, are very poorly known. The goal of the proposed research is to establish a reasonable model of the medullary pathways involved in controlling the level of the sympathetic outflow and in generating the basal sympathetic tone. Our long-term interest in the pathology of blood pressure regulation is the reason for chosing the rat as experimental subject because of the unique research opportunities presented by the availability of well-studied models of hypertension in this species. The starting point of the proposed investigation is a neurophysiological study of the anterior ventrolateral reticular formation since there is now overwhelming evidence that this area is essential in the maintenance of the sympathetic tone, in the operation of baroreceptor reflexes and in relaying sympathoexcitation due to the activity of higher structures. The proposal is also predicated on our preliminary results which indicate that this area may indeed contain sympathetic premotorneurons (projecting to the thoracic sympathetic nucleus) controling the sympathetic outflow to the vasculature. A large part of the work will therefore be devoted to understanding the role of these neurons in generating the sympathetic tone and their contribution to baroreceptor and other sympathetic reflexes. The nature of the neurotransmitters used by these cells will also be investigated. A second part of the study will be devoted to the afferent inputs of these cells and more particularly to those which convey their powerful baroreceptor inhibition. Finally, an attempt will be made to understand in neurophysiological terms the pathology of a model of human essential hypertension, the SHR rat. This work represents an extension of our previous research which was more specifically targeted toward understanding the role of central monoaminergic pathways in central autonomic regulations.

The regulation of regional blood flows and cardiac output is closely coordinated with respiration to ensure proper oxygen and carbon dioxide exchange in all body organs in the face of variable energy demands. Neural cardiovascular and respiratory reflexes which play important regulatory roles during health, may also contribute to clinical disorders such as sleep apnea in the adult and central and obstructive apnea of the newborn and the sudden infant death syndrome. The neuronal basis of respiratory rhythm generation and central cardiovascular control are only partially understood and the central connections responsible for the integration of these two essential functions remain largely conjectural. For example, the central pathways responsible for the excitatory influence of the medullary respiratory generator on the sympathetic outflow are totally unknown as are the intramedullary pathways responsible for the vasomotor effects produced by activation of cardioplumonary and other receptors which also control respiration. Much evidence already suggests that the rostral ventrolateral medulla may play a crucial role in cardiorespiratory integration and central chemosensitivity. Thus a major portion of the proposed research will be devoted to a study of several types of ventrolateral medullary cells which will be extensively characterized from the standpoint of connectivity, neurotransmitter content and electrophysiological properties. Efforts will be mainly concentrated on four rostral ventrolateral medullary reticulospinal cells, the C1 adrenergic neurons, neurons of nucleus interfascicularis hypoglossi, the Boetzinger complex expiratory related neurons and the sympathoexcitatory pace- maker neurons previously described in this laboratory. The experimental approach will include modern track-tracing techniques and single-unit recordings with iontophoretic application of drugs "in vivo" and "in vitro". Other experiments are also designed to test the hypothesis that certain types of bulbospinal respiratory premotorneurons located outside the rostral ventrolateral medulla might directly innervate spinal preganglionic neurons. In combination, these experiments will increase our understanding of the way in which central cardio- respiratory integration operates and may lead to the idenification of neurons with an essential role in central chemosensory function.

voltage /patch clamp

Mild hemorrhage produces autonomic and endocrine compensatory responses that are able to greatly minimize hypotension. These well- known responses are mostly caused by unloading of arterial barorceptors and include withdrawal of cardiovagal tone and a vigorous activation of the sympathetic outflow. If hemorrhage is more severe the autonomic compensatory mechanisms are paradoxically reversed. This second or ~ decompensatory~ phase of hemorrhage is characterized by bradycardia and a sharp decrease of sympathetic activity ( hemorrhage-induced sympathoinhibiton. HiSI) that produce further cardiovascular deterioration. Initiation of the decompensatory phase has been attributed to the activation of vagal afferent from the cardiopulmonary region however the central pathways of HiSI are not known. Nevertheless several studies have suggested that CNS opiod peptides may play a key role in genesis of this phenomenon. The objective of the proposed work is to investigate the CNS portion of the circuitry involved in hemorrhage induced sympathoinhibition and bradyardia. The study will be divided into five parts. Aim 1: using an established anestheized rat model, we will determine whether HiSI can be explained by a reduction in the discharge rate of the vasomotor neurons of the rostral ventrolateral medulla. Aim 2: we will determine whether hypotensive hemorrhage activates CVLM GABAergic neurons that arborize in RVLM. We will also determine whether the CVLM region is necessary for HiSI to occur. Aim 3: we will determine whether hypotensive hemorrhage reduces central respiratory drive and whether this effect contributes to HiSI Aim 4: we will determine whether the release of opioid peptides in RVLM contributes to hiSl Aim 5: we will determine which CNS neurons are activated by hypotensive hemorrhage in conscious rats and whether they are opiodergic or GABAergic. Understanding the neurophysiological mechanisms triggered by hypotensive hemorrhage should lead to improved ability to manage this all too frequent clinical problem.

The proposed conference will be the seventh in a series of FASEB Meetings dedicated to the Neural Control of Circulation. This topic is of ever increasing interest because of its relevance to major pathophysiological problems such as dysrhythmias, cardiac pain, hypertension, autonomic hyperactivity after spinal cord injury, sleep apnea etc. The main objective of the planned conference is to allow scientists and clinicians investigators to focus on closely interrelated aspects of the neural control of circulation for a week. This conference should help to open new areas of research and to provide a meaningful reexamination of previously accepted ideas. The recent advances in cellular and molecular techniques are making their impact in this very complex area of integrative physiology. This was evident in many of the presentations at the 1994 and 1996 conferences. The next program will continue to have a solid representation in cellular and molecular techniques that are most likely to provide new breakthroughs in this branch of neuroscience. However, we will maintain a vigorous emphasis on integrative physiological approaches which provide essential concepts to interpret clinical problems and potential strategies for pharmacological intervention. The conference will be organized around 9 plenary sessions each consisting of 4 presentations with ample time for discussion in between. The following topics will be covered: cardiac pain/afferent mechanisms and central processing; autonomic dysfunction after spinal cord injury; neurogenic models of long- term arterial pressure control; patterned autonomic responses during fever; role of medullary raphe in cardiovascular and nociceptive modulation; cardiovascular regulation during sleep and arousal and links to cardiovascular disease; neural regulation of circulation during exercise; molecular and genetic approaches to the study of cardiovascular regulations; imidazoline receptors and their endogenous ligands in the control of blood pressure. The 37 proposed speakers have already formally agreed to participate. Poster sessions and a poster presentation by selected students will complete the formal schedule, the rest of the time being dedicated to informal interactions.

DESCRIPTION (provided by applicant): Respiration relies on the spatially and temporally coordinated contraction and relaxation of several muscle groups that control lung inflation and airway diameter. A network of neurons that also controls the cardiac output and regional blood flows elaborates these motor outflows. This network is largely confined to the brainstem and its integrity is essential for blood gas homeostasis and to match oxygen delivery to tissues with metabolic demand. A relatively small fraction of the brainstem respiratory neurons seems dedicated to generating the breathing rhythm whereas the rest of the respiratory network, called the pattern generator, elaborates the various motor outflows. The present project focuses on a portion of the ventrolateral medullary reticular formation that contains neurons (the pre-Botzinger complex; pre-BotC) thought to be essential for the genesis of the respiratory rhythm and its control by central chemoreceptors and hypoxia. The cellular components of the pre-BotC are largely uncharacterized save for some of their neurophysiological properties in neonate rodents in vitro. The goal of the present project is to examine the functional neuroanatomy of the region of the pre-BotC in the adult rodent to further understand the cellular and molecular basis of respiratory rhythm generation and central chemoreception. This objective will be accomplished using a variety of neurophysiological and neuroanatomical experiments designed to identify the phenotype (main inotropic transmitter, selected peptides, receptors and channels) and connectivity of neurons whose discharge pattern and location will be fully characterized. The respiratory network is an essential life-sustaining aspect of brain function. The proposed work will contribute to understanding its basic cellular organization. The work represents the foundation upon which improved medical management of many cardiorespiratory pathologies may be eventually based. These pathological states include sleep apnea and its cardiovascular consequences, the bradycardic hypoxic syndrome of the neonate, infant death syndrome and, possibly, various pulmonary diseases, hypertension and congestive heart failure.

DESCRIPTION (provided by applicant): The central neural mechanisms that maintain breathing and achieve blood gas homeostasis during sleep have considerable clinical relevance for congenital central hypoventilation syndrome (CCHS), central and obstructive sleep apnea (OSA), sudden infant death syndrome (SIDS) and the treatment of pain with opiates. Under the auspices of this grant, we have identified and extensively characterized a population of lower brainstem neurons (ccRTN neurons) that could be playing a pivotal role in the CO2-mediatedregulation of breathing. The activity of these neurons is exquisitely sensitive to CO2 in vivo due to the fact that they respond directly to changes in the surrounding pH and receive powerful input from the oxygen and CO2- sensing carotid bodies. Furthermore, we have shown that the ccRTN neurons provide a strong excitatory drive to the respiratory pattern generator (RPG) and that their absence eliminates RPG activity under anesthesia regardless of the level of CO2. Recent findings of major significance to CCHS suggest that the ccRTN neurons may be even more important for CO2 homeostasis in conscious mammals than anticipated. Without input from these neurons, breathing may not be possible during sleep. In 2006, we demonstrated that, in rodents, the ccRTN neurons express Phox2b, the defining mutated gene in CCHS identified in 2003. The cardinal signs of CCHS are day time hypoventilation, total sleep apnea and loss of breathing stimulation by CO2. In 2008/9, geneticists found that the ccRTN neurons are selectively absent at birth in a mouse model of CCHS. In other words, the input-output properties of the ccRTN neurons, as we understand them currently, seem precisely appropriate for these cells to contribute to CO2 homeostasis via breathing. The genetic disease, CCHS, suggests that these neurons are critical in this regard, especially during sleep. These possibilities are fascinating and deserve to be thoroughly tested, not merely for their importance in CCHS but for their control of breathing during sleep in normal animals. Accordingly, the main objectives of this renewal application are: a) to assess the importance of the ccRTN neurons for involuntary breathing during various states of vigilance, b) to determine how the ccRTN neurons control breathing, specifically which respiratory neurons they target, and c) to further examine how the activity of the ccRTN neurons is regulated with special focus on their control by serotonin, another major neurotransmitter involved in breathing. The results from the proposed experiments will help to determine whether selective stimulation of these neurons could offer a therapeutic opportunity when hypoventilation is detrimental to health, e.g. OSA. To address these issues we will continue using the large repertoire of neurophysiological and neuroanatomical methods that we have developed in prior years and we will make heavy use of a new lentiviral vector approach to manipulate ccRTN neurons selectively in vivo, a method that has allowed us to unleash the power of optogenetics for the study of cardiorespiratory control.

Any condition that threatens homeostasis such as tissue injury, hemorrhage, hypoxia, infection, hypothermia, or psychological stress elicits changes in autonomic nervous system (ANS) function and specific behaviors. These changes can be beneficial for survival if transient but pathogenic if sustained, as in hypertension. The main new idea tested is that C1 neurons, a group of catecholaminergic/glutamatergic neurons within the rostral ventrolateral medulla of all mammals, including humans, are a nodal point for expression of these stress responses. That is, C1 neurons function like a switchboard enabling the expression of a large variety of ANS responses to acute physical and psychological stresses mediated by both sympathetic and parasympathetic divisions the ANS. The proposed experiments will use innovative methods (e.g. intersectional genetics, optogenetics, pharmacogenetics) designed to test key aspects of this theory, with particular emphasis on such novel topics as anti-inflammation, the prevention of hypoxic tissue damage and behavioral effects elicited by C1 cell activation. This knowledge is central to our understanding of how blood pressure and other physiological functions are regulated by the autonomic nervous system in healthy, diseased or stressed states. The specific aims tested are: 1: Is C1 cell stimulation sufficient to elicit physiological and behavioral signs of stress? Do C1 cells produce these effects by releasing glutamate or a catecholamine? Activation of C1 cells with optogenetics in unanesthetized transgenic mice is expected to result in signs of stress. 2: Are C1 cells necessary for responses to physiological or psychological stressors? Using loss of function optogenetics (archaerhodopsin) C1 cell activation is expected to be necessary to maintain BP and HR in awake rats subjected to non-hypotensive hemorrhage, hypoxia or bacterial infection. We will also test whether C1 cells are required for the autonomic (e.g. cardiovascular, respiratory, GI and anti-inflammatory components) and behavioral manifestations of restraint stress in conscious mice by either inhibiting (pharmacogenetically) or selectively destroying these neurons. 3: Which C1 cells regulate blood pressure? Using mouse intersectional genetics and Boolean vectors, subgroups of C1 cells defined by peptide expression or CNS projection will be transduced to selectively express ChannelRhodopsin2. These subgroups are expected to have distinct projections and their optogenetic activation is expected to elicit highly specific physiological and behavioral responses.