Shaun F. Morrison - US grants

Recent research on central cardiovascular control indicates the existence of a population of neurons in the lateral medullary reticular formation that provides a final common pathway conveying the excitatory drive to the spinal sympathetic nucleus that is critical for the maintenance of basal sympathetic tone. As described in the proposal, the characterization of these neurons will provide new information on the central neural mechanisms underlying the sympathetic control of blood pressure. The proposed studies will focus on the hypothesis that neurons in the rostral ventrolateral medulla (RVL) that contain the adrenaline-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT) mediate the tonic bulbospinal excitation of sympathetic preganglionic neurons (SPN) necessary for the support of resting blood pressure. Extracellular recording and antidromic activation techniques are combined with baroreceptor reflex activation and computer-aided analysis to identify sympathoexcitatory neurons in RVL that project to the sympathetic intermediolateral nucleus. Immunocytochemical staining is used to determine the proximity of the recording sites of these neurons to the locations of neurons containing PNMT (C1 cell group). Additional experiments are designed to examine (1) the baroreceptor reflex pathway providing an inhibitory control of the activity of these cells and (2) the activation of RVL sympathetic neurons from hypothalamic pressor sites. Microiontophoretic application of antagonist drugs will be used to classify the transmitter system(s) mediating the effects of RVL activation on the activity of SPNs. Together these data would not only increase our knowledge of the basic neural mechanisms involved in central blood pressure regulation, they might also provide a basis for (1) the evaluation of the role of altered neural function in hypertensive disease and (2) the design of pharmacologic approaches to antihypertensive therapy. The former possibility is tested in a final series of experiments to determine if the sympathetic hyperactivity of the spontaneously hypertensive rat is associated with increased activity of the sympathoexcitatory neurons in the C1 region of the RVL.

Blood pressure is regulated to a significant degree by the level of sympathetic nerve activity to the blood vessels. Vasoconstrictor sympathetic preganglionic neurons (SPNs), the source of this activity within the spinal cord, are located within a functionally heterogeneous population of SPNs whose discharge is determined primarily by descending inputs from a few discrete medullary regions. The long-term goal of the proposed research is an understanding of the spinal processing of information in these descending sympathetic pathways to SPNS, particularly with regard to those inputs to SPNs regulating vasoconstriction. Complementary electrophysiological, pharmacologic and electron microscopic studies are planned which examine the functional and structural relationships between splanchnic SPNs and their inputs from the rosaw ventral medulla and the caudal mphe nuclei. Specifically, criteria will be developed to distinguish splanchnic SPNs in the rat which regulate adrenal medullary secretion, visceral vasoconstriction, and gastrointestinal motility. Differences in the responses of these functional classes of SPNs to medullary stimulation will be examined to test the hypothesis that functional specificity exists within descending pathways to splanchnic SPNS. Iontophoretic application of transmitter agonists and antagonists will be used to determine the role of excitatory amino acid transmission in the brainstem stimulus-evoked excitations of SPNs and the potential modulatory functions of medullary catecholaminergic and serotonergic inputs to SPNS. Electron microscopic studies of the inputs to SPNs will combine Phaseolus anterograde labeling of the axon terminals of medullospinal pathways with immunocytochemical detection of transmitter markers to obtain information on the ultrastructural basis of the interactions between neurons in the medulla and those in the intermediolateral nucleus, including SPNs. This integrated approach will provide new information on the functioning of critical cardiovascular regulatory pathways to the spinal cord and may suggest new pharmacologic approaches to controlling arterial pressure.

Release of catecholamines from the chromaffin cells of the adrenal medulla into the circulation plays a significant role in mediating the cardiovascular and autonomic changes that support the behavioral and physiologic responses to stresses that not only challenge the maintenance of homeostasis directly, such as hemorrhage, hypoxia, cold, pain, hypoglycemia or severe exercise, but also those in which the challenge is only perceived, such as fright, anger, danger, anxiety or emotional stress. The long term goals of this research are (1) to elucidate the elements and organization of the central neural pathways that specifically regulate adrenal medullary functions, (2) to increase our understanding of the behavioral properties and the neurotransmitter actions within this unique central autonomic circuit and (3) to use the network controlling adrenal epinephrine release as a model to determine the mechanisms that provide selective central control of functionally- specific sympathetic outputs and that regulate the sensitivity of different sympathetic effector systems to common reflex inputs. In the proposed research, electrophysiologic and pharmacologic approaches will be used in anesthetized rats (1) to test the hypothesis that sympathetic preganglionic neurons (SPNs) regulating the adrenal secretion of epinephrine constitute a unique population of splanchnic SPNs that can be distinguished from other SPNs by their characteristic responses to physiologic stimuli that alter adrenal medullary epinephrine release, (2) to determine how populations of brainstem neurons with demonstrated projections to adrenal SPNs, influence the discharge of the SPNs controlling epinephrine release and which spinal neurotransmitters are involved in mediating these responses, and (3) to determine the supraspinal components of reflex pathways mediating the responses of adrenal SPNs to (a) shifts in blood pressure (b) alterations in blood glucose, and ~ chemoreceptor activation. Finally, simultaneous brainstem and spinal cord unit recordings will be made to provide direct evidence for the hypothesis that adrenal medullary-specific, sympathoexcitatory neurons in the rostral ventrolateral medulla mediate the reflex responses and tonic excitatory drive to adrenal SPNs controlling epinephrine release. These studies will contribute new information on the organization and control of central sympathetic networks and will increase our understanding of the central mechanisms mediating a specific aspect of the cardiovascular responses to stress, injury and environmental challenge. The results could have implications for the control of such responses that are likely to arise during surgery or in disease conditions such as Raynaud~s diabetes, hypertension, cardiac failure and sepsis.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Balancing energy expenditure with energy intake, metabolic requirements and energy storage is critical for maintenance of body weight, for sustained responsiveness to metabolic signals such as glucose, insulin and leptin and for homeostatic regulation of many body systems. Our experiments are defining and testing the hypothesis that energy expenditure in BAT is a potential sink for the consumption of surplus energy intake to prevent excess energy storage in white adipose tissue. Defining energy afferent signaling pathways, as well as those efferent neural pathways controlling the BAT energy expenditure effector will contribute to our understanding of the regulation of body weight and of energy substrate availability and to the design of therapeutic strategies to control disturbances in body energy homeostasis that result in diseases such as obesity, diabetes and hypertension.

The long-term objective of this research is to improve our understanding of the neural circuits regulating body temperature and how their function is altered during the fever response. Fever is a component of the acute-phase response to inflammatory stimuli. In uncontrolled conditions, it can threaten cellular homeostasis and survival. Treating the dysregulation of body temperature during fever will be aided by an understanding of the functional organization of the neural pathways that are activated by pyrogens and of the neurotransmitters mediating their effects. The proposed research plan will test the hypothesis that a population of neurons in the raphe pallidus (RPa) constitutes the sympathetic premotor pathway that is necessary for the activation of the sympathetic outflow to brown adipose tissue (BAT) in response to icv. PGE2, an intermediary in the "fever cascade". These data will provide the cornerstone for further investigations to determine (a) the pathways by which hypothalamic neurons regulating thermal homeostasis activate sympathetically-regulated thermogenesis during fever and (b) the principal neurotransmitters and receptors that regulate the activity of RPa thermogenic neurons during fever. The proposed studies to elucidate the function and pharmacology of the longitudinally-organized core pathway for the regulation of thermogenesis in BAT will use electrophysiological recordings from the sympathetic nerves to BAT and from single neurons in the medulla in combination with microinjection techniques to activate and interrupt neuronal activity in restricted regions of the neuraxis. The first specific aim will determine the role of RPa neurons in the increases in BAT SNA and BAT heat production evoked by icv. PGE2. The sympathetic premotor neurons that project to the spinal cord to mediate these effects will be identified and their responses to febrile stimuli will be determined. The second aim will identify the source of the tonic, GABAergic inhibition of RPa neurons and determine how this inhibition is modulated to produce fever-related increases in the sympathetic outflow to BAT. In the third aim, we will test the hypothesis that glutamate neurotransmission mediates the excitation of RPa neurons seen during the febrile response and that this excitation can be modulated by serotonergic inputs to thermogenic neurons in RPa. Understanding the pharmacology within pathways determining BAT sympathetic outflow will be a foundation for developing strategies to alter sympathetically- mediated heat production.

Description (provided by applicant): Fever is a defended elevation in body temperature that plays a significant role in the acute phase reaction stimulated by a cascade of endogenous pyrogens released during infection. The febrile increase in body temperature is the result of a patterned autonomic and somatic motor response orchestrated by the central nervous system in response to an increased production of the pyrogenic mediator, prostaglandin E2 (PGE2), in the preoptic area (POA), a principal thermoregulatory integration center in the brain. PGE2 binding to EP3 inhibitory receptors on neurons in the POA increases core body temperature by activating neural pathways to four principal thermoregulatory effectors: increased heat production from brown adipose tissue (BAT) thermogenesis, from shivering in skeletal muscle and from a marked tachycardia and increased heat conservation through cutaneous vasoconstriction (CVC). This same constellation of responses: augmented sympathetic outflows to BAT, to the heart and to skin blood vessels and increased somatic motorneuron discharge to muscle, also constitutes the cold defense homeostatic reflex response to stimulation of cutaneous cold receptors or falls in core temperature. In the previous funding period, we have made significant progress in understanding the functional organization and neurotransmitters regulating the activity in the thermoregulatory pathways mediating the increases in BAT thermogenesis, heart rate and CVC contributing to the febrile response to PGE2 in the POA and to cold defense responses to skin cooling. We propose to extend these studies by using the fruitful in vivo electrophysiological, anatomical and neuropharmacological approaches we have perfected over the past several years to address three specific aims that will provide new and important insights into the brain mechanisms effecting fever and performing the critical homeostatic function of thermoregulation. The first aim will test the hypothesis that somatic, as well as sympathetic febrile and cold defense responses are organized through a hierarchical pathway between the POA and the medullary raphe by determining the central neural mechanism underlying the PGE2- and cold-evoked shivering response. The second aim will determine the neural basis for the fundamental differences in performance between the thermoregulatory network regulating skin blood flow and that driving BAT thermogenesis. The third aim will focus on the key integrative neurons in thermoregulation: the output neurons of the POA, to understand the mechanism for the differential control of thermoregulatory effectors and to determine their role in mediating effector responses to neurotransmitter systems implicated in conditions of thermal dysregulation. PUBLIC HEALTH RELEVANCE: Understanding the central neural mechanisms mediating fever and cold defense is relevant to the development of therapeutic approaches to combat life-threatening excessive fevers (as during sepsis, toxemia, meningitis, some cancers) and to the management of the effects of thermal dysregulation that occurs during a variety of other clinically significant conditions such as cerebral ischemia and stroke, the abuse of amphetamine-based drugs, the hot flashes accompanying menopause and prostate surgery and the hypothermia induced during surgical anesthesia.

? DESCRIPTION (provided by applicant): The process of metabolizing fat and glucose to produce heat is unique to brown adipose tissue (BAT) (as well as beige adipose tissue), and is under the control of the CNS-generated sympathetic nervous system input to BAT. Although our research has defined the fundamental neural pathways through which thermal and febrile stimuli elicit changes in the sympathetic outflow to BAT, little is known about the neural circuits involve in the inhibitory influences on BAT activity, particularly those arising from sensors of metabolic signals. The findings of a consistent deficit of cold-activated BAT in obese humans and of marked improvements in glucose homeostasis upon BAT activation in models of obesity and diabetes are consistent with an over-active inhibitory regulation of BAT activity in the setting of metabolic disease. The new information from this research could contribute to reversing the detrimental inhibition of BAT activity and improve the outcomes for those with metabolic disease. In the proposed research project, we will determine the neural circuits through which viscerosensory, primarily metabolic, afferent information conveyed via the vagus and glossopharyngeal nerves produces an inhibition of the sympathetic outflow to BAT. Additionally, we seek to understand the roles of CNS regions, including the nucleus of the solitary tract, the ventrolateral medulla and the paraventricular hypothalamus, whose activation can inhibit BAT activity, in the overall inhibitory regulation of BAT activity. We propose a detailed series of in vivo electrophysiological, anatomical, neuropharmacological experiments to address four specific aims that will provide new insights into the inhibitory regulation of BAT thermogenesis. The first aim will address the functional organization of the CNS circuits through which vagal viscerosensory afferents impinging on the NTS can inhibit BAT activity, and will include studies on the region(s) and neurotransmitter systems within the NTS, and localize the likely visceral sources and the classes of relevant physiological stimuli that mediate the potent inhibitory regulation of BAT activity mediated via vagal viscerosensory inputs. We will pursue the pathway between the secondary sensory neurons in NTS and the BAT sympathetic preganglionic neurons in the spinal cord, whose reduced discharge ultimately mediates declines in BAT activity. The second aim will address the functional organization of the CNS circuits through which arterial chemoreceptor afferents in the glossopharyngeal nerve can drive a potent inhibition of BAT activity. In the third aim, we will characterize the role of the several populatins of neurons in the VLM in the inhibitory regulation of BAT activity, including their potential rolesin the BAT inhibitions elicited by vagal, arterial chemoreceptor and PVN activations. The fourth aim will determine is the functional organization of the CNS circuits through which activation of PVN neurons inhibits BAT activity.