Jack L. Feldman - US grants

This proposal is to study an aspect of the neural control of breathing in mammals. The long range goal of this laboratory is to explain the genesis and control of respiratory pattern in terms of the biophysical, synaptic and network properties of identified groups of neurons in the central nervous system (CNS). The specific goal of this project is to study pharmacological aspects of synaptic behavior of brainstem respiratory neurons. The interneuronal communications basic to the process of respiratory control by chemical mechanisms and mediated by, as yet unidentified, neurotransmitters. Identification of relevant neurotransmitters, the specific post-synaptic cells on which they act and the importance in understanding respiratory control and pattern generation., The basic premise of this proposal is that local administration of neurotransmitters which affect central respiratory drive, central chemoreceptor or peripheral respiratory afferent inputs, their agonists and/or antagonists will produce measureable, site-specific perturbations in the associated physiological processing. Thus, we propose to look at the effects on respiratory motor outflow and on the activity of individual respiratory neurons produced by perturbations such as alterations in arterial blood gases and/or afferent stimulation before and after ejection of transmitter related drugs in selected regions important in central respiratory control. By analyzing the alterations in respiratory neuronal activity induced by drug application, we will be able to identify the physiological role or the most likely candidates for respiratory related neurotransmitters. Transmitters which are likely to be involved in some manner of respiratory control include glutamate, glycine, GABA, substance P, the catecholamines and enkephalins.

This proposal is to study the neurobiological mechanisms underlying the transmission of respiratory drive to the diaphragm. Respiratory rhythm is generated in the brainstem, with the drive transmitted to spinal motoneurons via bulbospinal neurons, which have three target populations in spinal cord: I - Phrenic, intercostal and abdominal motoneurons; II - Propriospinal neurons in the upper cervical spinal cord; III - Segmental interneurons in the cervical and thoracic spinal cord. The relative importance of projections to each of these targets sites in determining the discharge of a given motoneuron is yet to be determined. We will focus on the monosynaptic projection to phrenic motoneurons. Our analysis will exploit the facile localization of bulbospinal respiratory neurons in the brainstem and of phrenic motoneurons in the spinal cord and their clear separation (greater than 20 mm in cat, greater than 10 mm in adult rat and greater than 2 mm in neonatal rat). The following questions will be addressed: 1 - What are the microanatomical, morphological and immunohistochemical characteristics of the descending monosynaptic projection to phrenic motoneurons? With neuroanatomical and immunohistochemical techniques, we propose to a) determine the neuromessengers localized within identified bulbospinal synaptic terminals; b) determine the neuromessengers localized in synapses of other inputs. 2 - What neuromessengers are released at the presynaptic terminals of descending bulbospinal neurons? We have preliminary evidence that an excitatory amino acid or related small peptide may be involved. What are the mechanisms of their pre- and post-synaptic action? These questions will be addressed in physiological and pharmacological studies in vivo and in vitro. This proposal represents a coordinated multidisciplinary approach, with descriptive studies providing the basis for specific experimental tests of factors influencing the behavior of phrenic motoneurons. Understanding the transmission of respiratory drive to motoneurons is of considerable importance in defining the mechanisms responsible for respiratory homeostasis and for pathologies where ventilatory failure results from the inability to generate the appropriate motor output. Moreover, we will be able to characterize in detail the relationship between a population of motoneurons and the premotoneurons providing their rhythmic drive. We will benefit greatly in this analysis by the physical separation of these populations and the ability to readily isolate each one from the other. Thus, we should be able to provide information relevant to the control of other important rhythmic motor acts such as locomotion mastication and nystagmus.

This proposal is to study an important aspect of control of the sympathetic nervous system, particularly as it may relate to cardiovascular control in mammals. We have developed a unique and powerful system for studying this problem that will allow the performance of studies difficult or impossible in other experimental systems. Sympathetic preganglionic neurons (SPNs) located in the intermediolateral (and intermediomedial) cell columns of the thoracic and lumbar spinal cord are the final common pathway for control of sympathetic activity. Their activity is a fundamental determinant of ganglionic activity, and ultimately of organ function, including the heart, vasculature, gut, sexual organs, etc. The goal of this project is to determine the factors that control SPN activity. Studies will be performed in a new and powerful in vitro experimental system, which consists of the brainstem and spinal cord. Under appropriate conditions, complex nervous system function is maintained, including generation of rhythmic respiratory activity and of rhythmic locomotor activity as well as activity in SPNs. Thus, we will perform our experiments under excellent conditions for pharmacological studies and with intact, functional neural networks. In particular, by altering the extracellular medium by addition of pharmacological agents affecting receptors, channels or transmitter action, or by ionic substitution while monitoring intracellular potentials under current- or voltage-clamp conditions, we will be able to obtain critical information concerning the intrinsic and synaptic properties controlling the behavior of SPNs. We will focus on the descending brainstem projections from the rostral ventrolateral medulla to SPNs, and will address the following questions. 1-What are the intrinsic properties of sympathetic preganglionic neurons that determine their response to synaptic inputs? 2-What neuromessengers are released at the presynaptic terminals of medullispinal neurons, including those of the rostroventrolateral medulla, onto spinal sympathetic preganglionic neurons? 3-What are the mechanisms of the pre- and post-synaptic action of these neuromessengers, as well as those of other possible transmitters or modulators acting at this synapse? Homeostasis in health, including cardiovascular homeostasis, depends critically on the transmission of sympathetic drive to SPNs, and it is important in the physiological responses to (cardiovascular) diseases of organ (cardiac or vascular) origin. Moreover, dysfunctions in central regulation of cardiovascular function may underlie such serious conditions as essential hypertension and stress-induced arrhythmias. Thus, understanding the neuropharmacology of this drive will be of considerable importance in defining the mechanisms responsible for cardiovascular homeostasis and for pathologies, such as neurogenic hypertension. Furthermore, the rational development of therapeutic drugs for cardiovascular problems that can be treated or ameliorated by manipulation of central autonomic activity affecting the heart and vasculature depends on such knowledge. In particular, if we can identify the neurotransmitter(s) transmitting sympathetic drive to SPNs, then drugs can be synthesized that can control their excitability. This could allow for precise control of tone of cardiac and vascular smooth muscle.

Understanding the central nervous system mechanisms controlling breathing in mammals represents one of the fundamental challenges in contemporary respiratory physiology. Establishing neural mechanisms at the cellular and synaptic level remains a central problem in unraveling the nature and function of the respiratory control system. This proposal exploits the unique properties of in vitro preparations from the neonatal rat nervous system to study basic cellular and synaptic mechanisms. We have developed novel in vitro slice preparation of the neonatal rat brainstem that contain functionally active respiratory networks and provide unparalleled access to neuronal elements critical for generation and transmission of respiratory rhythm under conditions that are optimal for analysis of cellular and synaptic processes. We propose a set of closely interrelated projects whose specific aims are to determine: (1) basic neuronal substrates for generation and transmission of respiratory rhythm in the neonatal nervous system; (2) cellular and synaptic mechanisms of rhythm generation, (3) neurochemical and synaptic mechanisms for modulation of rhythm, and (4) dynamical properties of respiratory neurons and networks by computational methods. The long-range goal of this multidisciplinary set of projects is to explain the ontogeny and neurogenesis of respiratory rhythm and pattern in mammals in terms of the biophysical, synaptic and network properties of CNS respiratory neurons. Information to be obtained from this proposal is fundamental for understanding basic human physiology and pathologies where ventilatory failure results from dysfunction of CNS mechanisms controlling breathing, including such diseases as apnea of prematurity, congenital central hypoventilation, central alveolar hypoventilation, and sudden infant death syndrome.

9451080 Beatty This proposal seeks funds to enrich the undergraduate neuroscience laboratory experience at UCLA by extending current exercises and experiments into the domain of the neuron and the genome. Two new neuroscience laboratories -the Interdisciplinary Neuroscience Laboratory and the Behavioral Neuroscience Laboratory -will benefit from the grant. The requested instrumentation will give students experience in intracellular microelectrode recording from cultured Aplysia neurons, the source of much basic knowledge of neuronal function; patch-clamp recording from PC-12 and N1E1-15 cells studying ion channels and receptors; and restriction analysis of DNA and DNA sequence analysis for identifying a series of clones that are expressed primarily in the brain. Patch-clamp and molecular biological methods will bc used together to study the properties of expressed receptors, channels, pumps, and other important membrane proteins in the Xenopus oocyte system. All these exercises are at the forefront of contemporary neuroscience and technically are well within the grasp of university undergraduates. Both the departmental and the interdisciplinary neuroscience laboratories are team taught, with each faculty member teaching a module in his or her own area of expertise. Up to date documentation and instructional material for all laboratory modules will be made available to all colleges and universities over Internet.

The mammalian brain is vigilant in control of breathing, regulating blood O2 and CO2 over a wide range of metabolic demand from birth till death. The brain makes efficient use of the respiratory musculature because the metabolic cost of continually breathing is considerable. In our attempt to understand the action of the brain in breathing, we ask two questions: How is respiratory rhythm generated? and, How is this rhythm transformed into a precisely modulated pattern of respiratory muscle activity? We address the first question in this proposal. In order to identify the neural mechanism underlying rhythmogenesis, one must first establish which neurons are involved. We hypothesize that neurons with pacemaker properties in the preBotzinger Complex (preBOtC) in the rostral ventrolateral medulla underlite rhythmogensis. The crux of testing the hypothesis that preBOtC pacemaker neurons are the kernel for generating respiratory rhythm (pacemaker hypothesis) is testing the causal role, if any, of pacemaker cells in rhythm generation. We propose to test the pacemaker hypothesis by: characterizing the ionic basis for bursting in pacemaker cells of the preBOtC. Determining if pacemaker neurons have the predicted responses to extrinsic stimuli that reset the respiratory phase timing. Determining if pacemaker neurons have the obligatory synaptic interactions if they are the source of respiratory rhythm. Determination of the mechanisms underlying breathing movements is basic to understanding human physiology and the pathophysiology of many diseases. Development of prophylaxis and treatment of such diseases as Sudden Infant Death Syndrome, apnea of prematurity, central alveolar hypoventilation, congenital central hypoventilation syndrome, sleep apnea and other forms of respiratory failure critically depend on such knowledge. Furthermore, the proposed work provides a unique exploration of an important integrative action of the brain. Exploiting a measurable behavior under controlled in vitro conditions permits novel experiments that can reveal important features in the link between synapses/neurons and behavior.

This proposal is to understand processes by which motoneurons innervating respiratory muscles function properly. Phrenic motoneurons integrate rhythmic, tonic and episodic inputs to produce an output that results in contraction of the diaphragm that subserves breathing, phonation, emesis, defecation, etc. Hypoglossal motoneurons control tongue muscles for swallowing, chewing, phonation and breathing, where they affect upper airway resistance. The aim of this application is to understand the control of the excitability of these neurons as they subserve breathing. Modulation of AMPA receptor function mediating inspiratory drive in these motoneurons by phosphorylation and dephosphorylation is postulated by a critical component in control of respiratory motor output. Electrophysiological studies will be done under in vitro conditions where we will record from neonatal rodent motoneurons while they receive endogenous respiratory-relative drive, conditions advantageous for determination of synaptic and cellular mechanisms specifically related to respiratory function. Histological studies will determine the presence within the phrenic and hypoglossal nuclei of kinases and phosphatases that can underlie phosphorylation of AMPA receptors of associated proteins. Pathologies of breathing such as sleep apnea, central alveolar hyperventilation, central inspiratory muscle fatigue and (perhaps) sudden infant death syndrome result from failure to generate adequate respiratory muscle activity; the degree that these failures occur at motoneurons is unknown. Therapeutic and/or abusive drugs that affect breathing, e.g., anesthetics or opiates, produce effects that may be ameliorated by pharmacological manipulation. Understanding the synaptic physiology of the control of breathing, in specific respiratory motoneurons, is essential for further rational development of therapies and treatments for breathing dysfunctions. Principles governing the control of respiratory rhythm generating neurons which also process rhythmic inputs mediated by AMPA receptors these. These results may reveal modulatory mechanisms common to other motoneurons, particularly those controlling muscle involved in rhythmic activities (e.g., locomotion, mastication, and nystagmus). The properties of different neuron types may be regulated phenotypically to optimize neuronal performance based on function. This proposal will provide the basis for such an interpretation concerning phrenic and hypoglossal motoneurons. Our unique advantage of making measurements in the context of behavior may reveal critical elements underlying control of neuronal excitability and its modulation in the normal transactions of the intact living brain.

[unreadable] DESCRIPTION (provided by applicant): Breathing is a remarkable behavior that regulates gas exchange to support metabolism and regulate blood pH. Failure to breathe properly in humans suffering from disorders such as sleep apnea, apnea of prematurity, congenital central hypoventilation syndrome, Rett Syndrome, central alveolar hypoventilation, and perhaps sudden infant death syndrome, leads to serious adverse health consequences, even death. If these pathologies are to be understood, the site(s) and mechanisms of respiratory rhythmogenesis must be revealed. Our laboratory, through support of this and related NIH grants, have contributed in important ways to our current understanding of the control of breathing. We made two novel discoveries in the previous grant period: 1. Partial lesions of the preBotzinger Complex (preBotC) in adult rats causes sleep disordered breathing. We will exploit this model of central sleep apnea to address an important question of considerable clinical relevance: What can be done to reduce the severity of central sleep apnea? Can drugs affecting serotonin, noradrenergic, cholinergic or specific peptide neurotransmission affect central sleep apneas when directly applied to the preBotC? If so, this will provide important information about possible causes of central sleep apnea and of the state-dependent role of the preBotC in generation of breathing pattern. 2. Respiratory rhythm generation appears to involve two distinct anatomically separated oscillators: the preBotC and the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG). We will look at mice (Krox20) with genetically-induced brainstem developmental anomalies that affect the RTN/pFRG. If our hypotheses are correct, we should see profound changes in expiratory-related activity. A complementary approach will be to record single neuron activity in the RTN/pFRG of rats. We predict we will observe rhythmically active RTN/pFRG when there is active expiratory motor output. These experiments, regardless of outcome, will help us to better understand RTN/pFRG function in normal and pathological states and under different experimental conditions and its interactions with the preBotC. The investigation and testing of the novel hypotheses arising from our recent discoveries should have fundamental impact on our understanding of breathing in humans in health and disease [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This grant will focus on determining the mechanisms underlying a form of activity-independent persistent changes in hypoglossal motoneurons in neonatal rats. We postulate that these mechanisms serve as a model for adaptive changes whose malfunction contributes to failures of respiratory plasticity, such as might underlie obstructive sleep apnea in humans. This work represents the convergence of three distinct aspects of brain function in mammals that are each of considerable importance in plasticity of the neural control of breathing: RESPIRATORY PLASTICITY- Changes in excitability of mammalian neurons lasting longer than a few minutes are the focus of considerable research. We will investigate a form of activity independent plasticity in a unique mammalian experimental system, a slice of brainstem from neonatal rodent that endogenously generates a rhythmic respiratory motor nerve output. We can therefore investigate mechanisms with all of the advantages of an in vitro system, yet in the context of the ongoing behavior. RESPIRATORY MOTONEURON FUNCTION - Motoneurons transform the internal actions of the brain into behavior, translating patterns of interneuronal activity into commands for skeletal muscle contraction and relaxation. How respiratory motoneurons respond to their inputs, and how their responses are regulated is basic to understanding how the brain maintains respiratory homeostasis. Motoneurons have the distinct advantage as model neurons in that their outputs are well-understood, i.e., muscle contraction, and in many cases, such as with inspiratory drive for breathing, their inputs are also well-understood. REGULATION OF BREATHING PATTERN-The mammalian brain is vigilant in control of breathing, regulating blood oxygen and carbon dioxide over a wide ranges of metabolism, posture and body movements, and compromises in muscle or cardiopulmonary function. Failure of the brain to maintain an appropriate motor output in humans suffering from disorders such as sleep apnea, apnea of prematurity, congenital central hypoventilation, central alveolar hypoventilation, and perhaps sudden infant death syndrome, leads to serious adverse health consequences, even death. If these pathologies are to be understood, the mechanisms controlling the output of respiratory motoneurons needs to be determined.

This award is for the development of a single-photon-sensitive confocal microscope, capable of true 4D (four dimensional: x, y, z, t) live imaging at video rate. It is based on an Image Intensified CMOS sensor (ICMOS) and a high-speed confocal scanner, which are designed to meet the following specifications: 1) High-speed (< 1 ms/frame), mega-pixel imaging with single-photon sensitivity. This is the same sensitivity as an EMCCD (Electron Multiplying CCD) but at one hundred times faster frame rate. 2) High-speed (< 1 ms/frame) confocal scanning for a single x-y focal plane. In addition, capability to scan in depth (z) up to 100 microns in 30 msec, resulting in a ''true 4D movie'' at video frame rate. 3) High-speed (< 10 ms/frame) FLIM (Fluorescence Lifetime Imaging Microscope) with < 100 psec lifetime resolution for a single x-y focal plane. This is a similar lifetime resolution as conventional scanning confocal microscopes but with a frame rate that is one hundred times faster. It will enable a ''true 4D FLIM movie'' at video frame rate. 4) Video-rate (~30 ms/frame) FLIM with true spectrum analysis for a single x-y focal plane. This new microscope may revolutionize the way millisecond time-scale phenomena are visualized in all biological systems, spanning from single molecules, single cells, and neural networks (such as the brain), to in vivo imaging of tissue in animals.

In addition to the scientific benefit of this new microscope, this award will contribute to multi-disciplinary education of students, at both the graduate and undergraduate level, at the forefront of biology, chemistry, physics, and engineering.

DESCRIPTION (provided by applicant): Project Summary This proposal aims to renew funding for a pre- and post-doctoral training program in NEURAL MICROCIRCUITS. The overall goal of this program is to train a cadre of outstanding researchers who will have: 1) an in-depth understanding of the principles that underlie the function of NEURAL MICROCIRCUITS in a broad range of nervous system function, and; 2) extensive research training in modern experimental approaches to analyzing NEURAL MICROCIRCUITS. Our program prepares trainees to conduct contemporary neuroscience research that bridges the gaps in understanding between synapses, single neurons, NEURAL MICROCIRCUITS and behavior. The program enhances basic pre- and postdoctoral training with an advanced NEURAL MICROCIRCUITS graduate course, a monthly journal club, and an annual DYNAMICS OF NEURAL MICROCIRCUITS symposium. In our inaugural funding period we have established a vigorous training program where new techniques, findings, and ideas are freely exchanged among the faculty, postdoctoral fellows and predoctoral students that fosters interaction and collaboration. Our success to date in achieving our ambitious training goals results from an outstanding pool of applicants and the strength of the interactive program faculty in a broad range of areas relevant to NEURAL MICROCIRCUITS. An outstanding research environment and excellent facilities are available at UCLA, with 24 faculty with highly active research programs with state of the art laboratories funded by significant extramural support. A broad range of core facilities are available that offer assistance, such as in molecular biology including in the making of viruses and of transgenic mice and advanced optics. This training program provides unique research training in NEURAL MICROCIRCUITS, which is fundamental for an understanding of the function and behavior of the CNS and for the development of therapeutic strategies for the treatment of pathological changes in the CNS. ! PUBLIC HEALTH RELEVANCE: Project Narrative Following training in the NEURAL MICROCIRCUITS program, predoctoral students and postdoctoral fellows will be uniquely poised to investigate and ultimately understand the NEURAL MICROCIRCUITS that are a critical link in understanding how behavior results from neurons and their interconnections. Such knowledge is essential to explain how nervous system disease results from neuronal dysfunction.

DESCRIPTION (provided by applicant): Delineating neurons that underlie complex behaviors is of fundamental interest. We will explore a novel method for extremely rapid changes in excitability of genetically targeted neurons to affect a robust and vital ongoing regulatory behavior in rodents, i.e., breathing. Breathing is a remarkable behavior that mediates gas exchange to support metabolism and regulate pH. A reliable and robust rhythm is essential for breathing in mammals. Failure to maintain a normal breathing rhythm in humans suffering from sleep apnea, apnea of prematurity, congenital central hypoventilation syndrome, hyperventilation syndrome, Rett syndrome, and perhaps sudden infant death syndrome, leads to serious adverse health consequences, even death. Various neurodegenerative diseases, such as Parkinson's disease, multiple systems atrophy and amyotrophic lateral sclerosis, are associated with sleep disordered breathing that we hypothesize results from the loss of neurons in brain areas controlling respiration. If breathing is to be understood in normal and in pathological conditions, the mechanisms for respiratory rhythmogenesis must be revealed. We focus on a brain site essential for generation of the normal breathing pattern, the preB"tzinger Complex. Using a viral delivery system, we will express genetically encoded rhodopsin-like molecules in various phenotypes of neurons in the preB"tzinger Complex. Rapid changes in excitability of these neurons by administration of light pulses delivered via an optical fiber implanted in the preB"tC in anesthetized, awake or sleeping rats should produce noticeable, even profound perturbations in breathing. Analysis of such perturbations will provide an extraordinary window into understanding mechanisms of respiratory rhythm and pattern generation. PUBLIC HEALTH RELEVANCE: In humans, continuous breathing from birth is essential to life and requires that the nervous system generate a reliable and robust rhythm that drives inspiratory and expiratory muscles. The proposed studies will significantly advance our understanding of the neural mechanisms generating respiratory rhythm and shed light on human disorders of breathing.

DESCRIPTION (provided by applicant): We study the generation and regulation of respiratory rhythm in mammals. Breathing is a remarkable behavior in vertebrates that regulates gas exchange to support metabolism. A reliable and robust rhythm is essential for breathing in mammals. The failure to maintain a normal breathing rhythm in humans suffering from disorders such as sleep apnea, apnea of prematurity, congenital central hypoventilation syndrome, central alveolar hypo- ventilation syndrome, hyperventilation syndrome, Rett syndrome, and perhaps sudden infant death syndrome, leads to serious adverse health consequences, even death. Various neurodegenerative diseases, such as Parkinson's disease, multiple systems atrophy and amyotrophic lateral sclerosis, are associated with sleep disordered breathing that we hypothesize results from the loss of neurons in brain areas controlling respiration. If breathing is to be understood in normal and in pathological conditions, the site(s) and mechanisms for respiratory rhythmogenesis must be revealed. PreB"tzinger Complex (preB"tC) neurons in the brainstem underlie respiratory rhythm generation in vitro and are essential for breathing in awake adult rats in vivo. While our ultimate goal is to explain the generation of respiratory rhythm in intact mammals, in particular humans in health and disease, studies necessary to examine basic cellular/network mechanisms are presently impossible under in vivo conditions. In this proposal we will exploit a validated and powerful in vitro model of breathing, the rhythmic medullary slice generating a respiratory-related motor output, to investigate cellular properties of preB"tC neurons and advance our understanding of respiratory rhythmogenesis. We will use state of the art techniques such as calcium imaging and calcium uncaging to test the hypothesis: Calcium ion transients play a critical role in determining the dynamic properties of preB"tC neurons that are widely accepted to play a necessary role in respiratory rhythmogenesis. We propose 2 SPECIFIC AIMS. AIM 1: We will determine the temporal and somatodendritic distribution of calcium transients in preB"tC neurons. AIM 2: We will determine the effects of calcium transients on the dynamic properties of these key neurons. By detailing how intraneuronal calcium transients affect respiratory rhythm we will significantly improve our knowledge of neural control of breathing. These studies should make fundamental contributions to our understanding of breathing in humans in health and disease. PUBLIC HEALTH RELEVANCE: In humans, continuous breathing from birth is essential to life and requires that the nervous system generate a reliable and robust rhythm that drives inspiratory and expiratory muscles. The proposed studies will significantly advance our understanding of the neural mechanisms generating respiratory rhythm and shed light on human disorders of breathing.

DESCRIPTION (provided by applicant): Delineating neurons that underlie complex behaviors is of fundamental interest. We will exploit a novel method for extremely rapid changes in excitability of genetically targeted neurons to affect a robust and vital ongoing regulatory behavior in rodents, i.e., breathing. Breathing is a remarkable behavior that mediates gas exchange to support metabolism and regulate pH. A reliable and robust rhythm is essential for breathing in mammals. Failure to maintain a normal breathing rhythm in humans suffering from sleep apnea, apnea of prematurity, congenital central hypoventilation syndrome, hyperventilation syndrome, Rett syndrome, and perhaps sudden infant death syndrome, leads to serious adverse health consequences, even death. Various neurodegenerative diseases, such as Parkinson's disease, multiple systems atrophy and amyotrophic lateral sclerosis, are associated with sleep disordered breathing that we hypothesize results from the loss of neurons in brain areas controlling respiration. If breathing is to be understood in normal and in pathological conditions, the mechanisms for respiratory rhythmogenesis must be revealed. We focus on two brain sites essential for generation of the normal breathing pattern, the preBvtzinger Complex and the retrotrapezoid nucleus/parafacial respiratory group. Using a viral delivery system, we will express genetically encoded opsins in various phenotypes of neurons in these key regions. Rapid changes in excitability of these neurons by administration of light pulses delivered via an optical fiber implanted in these sites in anesthetized, awake or sleeping rats should produce noticeable, even profound perturbations in breathing. Analysis of such perturbations will provide an extraordinary window into understanding mechanisms of respiratory rhythm and pattern generation. PUBLIC HEALTH RELEVANCE: In humans, continuous breathing from birth is essential to life and requires that the nervous system generate a reliable and robust rhythm that drives inspiratory and expiratory muscles. The proposed studies will significantly advance our understanding of the neural mechanisms generating respiratory rhythm and shed light on human disorders of breathing.

The CSORDA Pilot Program was established in 2002 and has proven to be an important mechanism for evolution of the Center by incorporating new and innovative project areas and technical expertise. We are proposing a modest program with $40,000 available each year to fund two Pilot Projects. Logistical management of the Pilot Program is the responsibility of the Administrative Core. The selection process Involves a call for proposals and analysis of the proposals by an ad-hoc Pilot Project Selection Committee chaired by Dr. Edythe London. The Pilot Committee, with input from the Advisory Board recommends projects for funding, which are subsequently presented by the Pilot Project PI to all Center personnel at one of the bi-weekly Center meetings. This process provides the opportunity for open critique and optimization of Pilot Projects by all members of the Center, and often develops productive collaborative projects between CSORDA and Pilot Project Pi's. Final decisions for funded Pilots are made at the Steering Committee meeting, with the Directors making decisions in consultation with Dr. London and the Advisory Board if consensus cannot be reached. The Administrative Core provides a mechanism for budgetary and scientific oversight of the pilots. Choice of funded pilots will be made according to the innovation and excellence of the research proposed, as well as, its likely impact in the area of substance abuse research. Priority will be given to Projects that could have high impact, those most closely related to the theme of the Center, and those that may offer new technologies and research for future incorporation into CSORDA. Two Pilot Projects have been selected for the first year of funding: Dr. Jack Feldman will use the conditional Mu knockout mice (developed in the laboratory of Dr. Kieffer) to assess cell-types in the preBotzinger Complex that are involved in opioid-induced respiratory depression, and Dr. Hongwei Dong will assess connectivity between different brain areas involved in opioid reward.

PROJECT SUMMARY Breathing is a remarkable behavior fundamental to life that mediates gas exchange to support metabolism and regulate pH. A reliable, non-stop, robust rhythmic pattern of respiratory muscle activity is essential for breathing in mammals. Failure to maintain a normal breathing pattern in humans suffering from sleep apnea, apnea of prematurity, congenital central hypoventilation syndrome, hyperventilation syndrome, Rett syndrome, and perhaps Sudden Infant Death Syndrome, leads to serious adverse health consequences, even death. Various neurodegenerative diseases, such as Parkinson's disease, multiple systems atrophy, and amyotrophic lateral sclerosis, are associated with sleep disordered breathing that we hypothesize results from the loss of neurons in brain areas controlling respiration. If breathing is to be understood in normal and in pathological conditions, the mechanisms for respiratory central pattern generation must be revealed. We focus on two brain sites essential for generation of the normal breathing pattern, the preBötzinger Complex and the retrotrapezoid nucleus/parafacial respiratory group. We propose a broad series of experiments both in vivo and in vitro in rodents using advanced techniques including: viral delivery to express genetically encoded opsins or DREADDs in key subpopulations of neurons in these regions; advanced optical techniques to determine the contributions of the preBötzinger Complex microcircuit to rhythm generation; state-of-the-art neuroanatomical techniques to establish, in appropriate and necessary detail, the interconnectivity of the brainstem respiratory pattern generator. The data from these experiments will provide an extraordinary window into the mechanisms underlying respiratory rhythm and pattern generation.

Neural basis for positive effects of breathing on emotional state Principal Investigator: J.L. Feldman Co-Investigator: M. Fanselow ABSTRACT: Over the past few decades, research involving the neural control of breathing has centered on how the breathing rhythm is generated and how sensory systems detect internal and external changes to modulate the breathing pattern. While we have learned that the regulatory breathing rhythm originates in the medulla, we know little about the neural circuits that are involved in volitional and emotional control of breathing and the mechanisms by which controlled breathing affects emotional state. The positive effects of controlled breathing on emotional state have been observed across many contexts and in the clinic. Using objective behavioral, physiological, and neuroanatomical parameters, we propose to establish the neural pathways from the brainstem central pattern generator for breathing to suprapontine regions that effect and/or affect emotional state and develop a standard protocol in rodents to measure the effects of changing breathing on emotional state. Development of such an animal model will allow us to probe in behaving rodents the neural circuits that are involved in reducing stress, anxiety, pain perception, depression, etc., as a result of epochs of controlled breathing. The proposed research has the potential to lead to more effective methods for treating debilitating negative emotional states, including depression and panic.