Michael C. Andresen, Ph.D. - US grants

The role of baroreceptor inputs in the modulation of sympathetic nerve activity both under control conditions and under conditions known to alter baroreceptor characteristics will be examined. Sympathetic nerve activity will be studied at three different levels. In the first phase of the study, activity of single afferent fibers from sympathetic nerves will be measured in anesthetized rabbits. In the second phase activity from whole sympathetic nerves will be recorded in anesthetized rabbits. In the final phase chronic recordings of sympathetic nerve activity will be made in conscious, unanesthetized rabbits. The studies will primarily be concerned with renal sympathetic nerves but cardiac and muscle sympathetic nerves will also be included. In all phases ofthis study, surgically implanted perivascular balloons will be used to briefly raise and lower blood pressure to determine the relationship of sympathetic nerve activity to mean arterial blood pressure. Curve characteristics including the blood pressue at which activity is minimal, the slope on gain and the range o activity for the barofeflex relationship will be determined. Prolonged alteration of resting blood pressure will be used to study the effects of acute barcreceptor resetting on baroreflex modulation of sympathetic activity. Single fiber efferents will be examined to determine whether whole nerve activity reflects responses in a homogenous or heterogeneous population of individual efferents and thus to interpret the response of whole nerve sympathetic activity. The contribution of afferent input from high and low pressure baroreceptors will be studied by selective aortic and carotid sinus denervations. More specific knowledge of the baroreceptor involvement in modifying sympathetic outflow is important to understanding the neural control of the circulation during prolonged blood pressure alterations, such as duing therapeutic drug treatment.

Excessive salt-ingestion in man has long been associated with an increased risk of developing hypertension, yet the mechanisms by which salt acts are poorly understood. In the Dahl rat model, Salt-Sensitive rats (DS) develop elevated blood pressures on a high salt (HS) diet while Salt-Resistant rats (DR) remain normotensive. Recent research has suggested that there is a significant neurogenic contribution to the development of hypertension in DS rats. The arterial and cardiopulmonary baroreflexes of DS rats are depressed even before exposure to high salt and the subsequent development of hypertension. This project will test whether abnormal function of the cardiovascular mechanoreceptors contributes to these decreases in baroreflex sensitivity in DS rats. I will use two in vitro preparations to study quantitatively the response characteristics of aortic arch baroreceptors and cardiopulmonary mechanoreceptors located in the superior vena cava just outside the right atrium. This in vitro approach allows a much more precise control of experimental conditions including reproducible, well-defined pressure inputs and ionic environment. In addition, the geometry of the vessel walls in which these mechanoreceptors are found can be closely approximated by a simple right cylinder so that the contribution of vessel wall distensibility and mechanics can be assessed through measurements of vessel diameter. Single mechanoreceptors will be tested for steady-state discharge characteristics including threshold and suprathreshold sensitivity, for rapid resetting to changes in the conditioning pressure and for sensitivity to changes in external ionic concentrations. Of particular importance are the comparisons of an inbred, nonselected strain of control rats (Sprague-Dawley) to both DS and DR and the effect of low salt and high salt diets.

Baroreflexes are an important part of the neural regulation of the cardiovascular system, but the cellular basis of the central nervous system (CNS) contribution to these reflexes is poorly understood. The area of densest innervation by the baroreceptor afferents is in the dorsomedial portion of the nucleus of the tractus solitarius (NTS). Reflex studies and extracellular recordings of neuron activity from the medulla suggest that the NTS is a key area where incoming afferent information is transformed before going on to other brain areas involved in the control of heart rate and blood pressure. A number of studies suggest that the properties of NTS neurons may be altered during hypertension. The proposed work will focus on characterizing the cellular basis of synaptic transmission at the afferent-NTS synapse. CNS studies are hampered by difficulties in gaining physical access to these small neurons and in recording conditions in the intact brain. Thus most of what we know about NTS neurons is based on extracellular activity recordings which provide limited information. An in vitro longitudinal slice preparation of the rat medulla has been developed to study medial NTS neurons using intracellular recordings to measure responses to afferent synaptic input. The longitudinal slice offers important advantages by preserving both lengthy sections of the solitary tract and key anatomical landmarks for microelectrode placement and by providing precise of control experimental conditions such as drug concentrations. Electrical stimulation of afferent axons will be used to evoke postsynaptic responses. Several key issues in afferent-NTS synaptic transmission will be examined including: 1) the mechanism of frequency dependent depression of synaptic responses and the influence of intermittent or burst modes of stimulation, 2) the identity of the primary excitatory transmitter and its postsynaptic receptor type, 3) the potential role and identity of inhibitory transmitters in synaptic modulation, and 4) possible changes in primary excitatory transmission or its modulation in genetic hypertension. Primary candidate neurotransmitters include glutamate, substance P, gamma-aminobutyric acid, and norepinephrine. Dual marking (with transganglionic transport of horseradish peroxidase in aortic baroreceptor nerves and intracellular dye injection) will be used to identify neurons characterized electrophysiologically which received cardiovascular afferent inputs. Immunocytochemistry will identify neurotransmitters localized on cell bodies or processes. These studies should provide important new information concerning the CNS mechanisms involved in a critical step of reflex autonomic control of the hear and blood pressure during normal and pathological states such as hypertension.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Cardiovascular dysfunction is frequently observed during use of general anesthetics (GA), although relatively little is known about the cellular mechanisms or specific CNS sites affecting cardiovascular regulation. We will use two model in vitro systems to study anesthetic mechanisms in neurons of a major autonomic brainstem reflex, the cardiac baroreceptor reflex (BRX). A major overall hypothesis is that differences in GA actions on cardiovascular regulation results from selective actions at different brainstem sites. The present proposal tests important aspects of this general overall hypothesis by focusing on GA actions on nucleus tractus solitarius (NTS) and nucleus ambiguus (NA) neurons. A major overall goal of this proposal is complementary testing of NTS and NA for differential sensitivities to GAs. NTS and NA are stations in the core cardioregulatory parasympathetic reflex arc. In our dual team approach, we combine complementary expertise in our two labs. Our work is directed toward addressing key elements controlling the activity of NTS and NA neurons and evaluating their susceptibility to GA actions. The new proposed studies focus on the mechanisms by which isoflurane and propofol act within the BRX. The issues of the proposal focus on complementary targets within NTS and NA and we will build upon new understanding of these neurons arising from our previous funding period. Our novel approaches allow us to study NTS neurons receiving baroreceptor contacts and NA cardiac preganglionic parasympathetic neurons in unique brain slice preparations. Afferents differentiated by their axon class evoke very different reflex responses and preliminary findings suggest that these pathways differ fundamentally in the sensitivity of these NTS neurons to anesthetics. NTS studies will address important aspects of this new finding. Cardiac vagal NA neurons were found to be intrinsically silent and glutamatergic, GABAergic, and cholinergic inputs are key. Thus, our focus overall is on modulation of excitability and synaptic transmission. Three major potential broad cellular targets of anesthetics within the medulla include: excitatory synaptic transmission, inhibitory synaptic transmission, and voltage-gated ion channels. By examining different anesthetics, we may also better understand whether potentially new anesthetics might be designed to spare these cardioregulatory sites. [unreadable] [unreadable]

baroreflex; anesthetics; pharmacokinetics; brain stem; brain mapping; cardiovascular disorder; glutamate receptor; GABA receptor; efferent nerve; heart rate; ketamine; blood pressure; vasoactive agent; vascular smooth muscle nervous control; halothane; isoflurane; nitroferricyanide; muscarinic receptor; beta adrenergic receptor; neurotransmitter metabolism; laboratory rat;

[unreadable] DESCRIPTION (provided by applicant): Neural reflexes rapidly adjust the cardiovascular system yet little is established about mechanisms governing their central nervous system pathways. The initial neurons are located within nucleus tractus solitarius (NTS). NTS integrates primary afferent information with CNS inputs. Our Research Plan will provide a cellular understanding of NTS integration and autonomic control. Heterogeneity in neuronal phenotype and pathway may reflect inherent patterns of specificity and, ultimately, represent therapeutic opportunities. Our Research Plan will address questions regarding heterogeneity of cellular properties as well as pathways of network organization within and beyond NTS. Our approach capitalizes on knowledge of cranial sensory neurons to probe the identity of NTS neurons. Our major long-term goal tests the hypothesis that sensory synapses within NTS are sites of major transformation of information. Our work features a cellular electrophysiological approach to NTS utilizing unique in vitro preparations of brain stem slices and dissociated cells. This renewal proposal focuses on synaptic processing (pre- and postsynaptic) as well as defining particular pathways and subsets of NTS neurons. The combination of molecular/cellular phenotype together with pathway information will allow us to define the roles of particular neurons within pathways. In brain stem slices and dissociated neurons with intact native boutons, we use live cell imaging, electrophysiology including paired recordings, plus retro- and anterograde labeling. We will target key areas in NTS sensory processing: presynaptic mechanisms regulating transmitter release, transmitter interactions, and potassium channels. Our Aims focus on GABAb receptors at 2nd order NTS neurons, regulation of GABAergic NTS neurons, cell-cell coupling, mechanisms of vasopressin action, and PVN and CVLM-projecting neurons. These Aims will provide new and direct information about NTS that leads to a better understanding of the normal basis of the neural control of the circulation as pathophysiology. [unreadable] [unreadable] [unreadable]

Beat to beat regulation of the cardiovascular system depends on an intact baroreflex. This reflex arc first synapses in the nucleus tractus solitarius (NTS) where glutamate is released. Baroreflex function is compromised in common life threatening disease states: hypertension, shock, and heart failure. Work in large, accessible CNS terminals suggests that the presynaptic control of glutamate release involves ion channels and 2nd messenger systems that regulate vesicle exocytosis. Glutamate regulation differs across neurons, but the mechanisms are poorly understood. Reflex pathways regulating the cardiovascular and respiratory systems depend on brainstem neurons and these reflexes are initiated by cranial nerve primary afferents acting within the nucleus tractus solitarius (NTS). Little is known about how primary afferents behave centrally. The small size of these terminals has made direct investigation difficult. We have developed methods to permit patch clamp recording from single nerve terminals in NTS. Our Research Plan will use these methods to address our driving hypothesis that important mechanisms of regulation in NTS depend on the identity of the afferent neuron. Our Preliminary Work demonstrates that NTS neurons offer a unique opportunity because: 1. we can directly visualize, identify, stimulate and electrophysiologically record from single nerve terminals, 2. NTS receives pharmacologically distinguishable afferent terminals that form subclasses known to arise from molecularly distinct peripheral neurons, 3. Subsets of these terminals can be labeled to provide links to functionally distinct afferents. Our Plan encompasses the efforts of two labs with complementary expertise suited to this problem. We will use direct patch recording and stimulation of single terminals, as well as imaging, to study the mechanism of frequency dependent synaptic depression and peptide modulation of glutamate release. The work capitalizes on using markers of primary afferent terminals to distinguish the various sub classes (TRPV1 and P2X3;C- and A-type afferent terminals, respectively). Specific Aims concern differential sodium, potassium and calcium channel expression across A- and C-type cranial afferent synaptic terminals plus the presence of new presynaptic mechanisms regulating synaptic cleft calcium. The different sub classes of afferent nerve terminal will be also be compared in terms of their control of glutamate release and modulation by peptides.

DESCRIPTION (provided by applicant): Transient Receptor Potential Vanilloid Type 1 Receptors (TRPV1) contribute to detection of noxious heat (>420C) and tissue damage by spinal primary afferent nociceptors. This proposal will examine the mechanisms by which TRPV1 expression in cranial primary afferents within the solitary tract nucleus (NTS) control a newly discovered form of synaptic transmission - TRPV1 mediated asynchronous glutamate transmission. Our Preliminary Studies indicate that this TRPV1 mechanism is active at physiological temperatures and potentiates long-lasting glutamate release in an afferent activity-dependent fashion - the latter being a new form of synaptic plasticity. This new form of glutamate transmission is only present in transmission from capsaicin sensitive solitary tract afferents. The Research Plan proposes to establish the mechanisms of action of TRPV1 in ST afferent transmission with a focus on CNS function in cardiovascular control. Our global hypothesis proposes that TRPV1 localized to presynaptic cranial afferent terminals is a focal integrator of multiple signals in NTS. In this pivotal role, we postulate that TRPV1 serves as a gain rheostat - increasing or decreasing the impact of unmyelinated baroreceptor afferents. We will investigate the role of TRPV1 in NTS in combining signals related to temperature; G-protein coupled receptors, membrane derived lipid mediators and other signals. Preliminary work indicates that the asynchronous TRPV1 pool of excitatory glutamate vesicles is regulated independently from the synchronous glutamate vesicle pool responsible for excitatory postsynaptic currents triggered at low jitter latency by afferent action potentials - a synchronous release process that appears identical in all solitary tract afferents. My laboratory has extensive experience with TRPV1 mechanisms in peripheral baroreceptors, baroreceptor reflexes, and central ST transmission. Studies will rely on methods including electrophysiological, live cell imaging, dye tracing and reflex assays in a combination of rats, mice and transgenic mice. Our Specific Aims include evaluations of cannabinoid signaling in afferent activity-dependent generation of asynchronous glutamate release, protein kinase C requirements, G-protein coupled receptor contributions to sensitizing asynchronous release, and NTS TRPV1 impact on cardiovascular regulation. The proposed research will help us to fully understand the normal basis of these neural control mechanisms in order to identify pathophysiological changes and new therapeutic avenues in clinical syndromes that may include consequences of central nervous system inflammation, hypertension, stroke, metabolic syndrome, and heart failure - all of which display altered autonomic reflexes to detrimental effect.

Project Summary Transient Receptor Potential Vanilloid Type 1 Receptors (TRPV1) are present in the central terminals of cranial visceral afferents including arterial baroreceptors and airway afferents within the solitary tract nucleus (NTS) but the function of central TRPV1 is unclear. TRPV1 is a calcium channel with three separate ?openers? vanilloids, pH, and heat. Our recent cellular investigations demonstrate that TRPV1 calcium controls an independently regulated pool of glutamate vesicles that is distinct from the pool of vesicles released by action potentials. TRPV1-operated vesicles generate spontaneous excitatory synaptic events (EPSCs). Action potential evoked EPSCs are triggered by voltage activated calcium channels separately from TRPV1-operated glutamate release. Cooling to unphysiologically low temperatures 30°C suppresses TRPV1 release and vanilloid agonists sensitize thermal sensitivity. The present proposal stems from the observation that in animals fed a high fat diet (HFD), blockade of TRPV1 receptors within medial NTS reduces blood pressure and heart rate. Animals fed control diets do not respond to TRPV1 blockade suggesting an endogenous lipid agonist induced in NTS by the HFD. We will examine the mechanisms by which ST TRPV1 drives glutamate transmission normally and during exposure to a HFD. Our in vivo preliminary results suggest that HFD generates a vanilloid-like mediator which controls afferent triggered reflex function within NTS. The Research Plan proposes to establish the mechanisms of action of vanilloids in ST afferent transmission with a focus on CNS function in aortic baroreflex control. Our global hypothesis proposes that ST TRPV1 serves as a focal integrator of multiple signals in NTS with a primary reporting output of glutamate release. The Specific Aims will investigate whether TRPV1-operated glutamate activates metabotropic glutamate receptors on GABA release, whether postsynaptic depolarization modulates presynaptic TRPV1 mediated glutamate release, whether B-type GABA receptors alter myelinated baroreceptor transmission during HFD and how TRPV1 activation in NTS during HFD alters baroreflex responses. My laboratory has extensive experience with TRPV1 mechanisms in peripheral baroreceptors, baroreceptor reflexes, and central ST transmission. We will rely on methods including electrophysiological, live cell imaging, dye tracing and assays of whole animal reflex characteristics to understand TRPV1 function from cell to reflex. We will team with the Madden lab for whole animal assessments. The proposed research will help us to better understand the normal basis of these neural control mechanisms as well as identify pathophysiological changes and shed light on homeostatic control that include consequences for central nervous system inflammation, hypertension, stroke, metabolic syndrome, and heart failure to alter autonomic reflexes to detrimental effect.