Terry L. Powley, PhD - US grants

The proposed project continues a systematic investigation of the neural mechanisms of body weight regulation and feeding. Ample evidence now exists that the vagus nerve is importantly involved in body energy regulation and food intake. Currently, however, continued progress in analyzing the vagal mechanisms implicated in the behavior and physiology of ingestion is seriously hampered by a lack of basic information about the organization of the vagal nuclei in the brainstem, the distribution of the vagus in the abdomen, and functional patterns of vagal activity. Compounding the problem, the lack of the same information has also retarded the development of adequate tests of vagotomy or normal vagal function. The lack of these tests has, in turn, further impeded research on the precise roles of the vagus in weight regulation, feeding and related disease processes. The immediate goal is the continuation of a series of promising analyses, begun during the last period, that are delineating the organization of the vagus through the use of converging physiological, anatomical, surgical and behavioral techniques. Five sets of experiments designed to provide a foundation for later, more extended functional analyses are proposed: The first two sets consist of anatomical and physiological characterizations of the topographic organization of the vagal motor nuclei in the medulla oblongata. That information is then also applied in a high resolution test for different selective vagotomies. The next two sets employ similar experimental methods to characterize the functional organization and distribution of the individual branches of the vagus nerve in the abdomen. The fifth group of experiments, drawing on the results of the first four, includes a number of fine-grained analyses of the role of defined vagal mechanisms in the altered metabolism and food intake of different animal models. The program outlined will extend our understanding of major neural mechanisms implicated in metabolic and digestive diseases including obesity, diabetes, anorexia, vagal dysfunction, peptic ulcers, and eating disorders.

The present proposal requests support for an optical sectioning and extended 3-dimensional reconstruction microscopy system to support life sciences research at Purdue University. This installation is to consist of a confocal scanning laser microscope integrated with a complementary morphometry/image analysis soft- and hardware package. The system will be organized as a separately housed independent facility, the Purdue University Optical Sectioning and 3-D Reconstruction Laboratory. In addition to operating as a self- contained stand-alone laboratory, the proposed facility will also be linked directly, by an existing campus computer network, to the Purdue University Computing Center, to all University users' laboratories that are on the network, and, indeed, to other research facilities nation-wide through the same computer network. The proposed system will serve an extensive users group of 16 investigators (as well as their research associates and students) representing 7 different departments at Purdue and directing a very significant amount of NIH supported research (over $3 million for the current year). As a group, these users have extensive experience with, and rely heavily on, standard epi-fluorescence microscopy; they also have extended needs for 3-D reconstruction capabilities in their microscopy. The requested equipment will enhance and extend these research programs through the application of the revolutionary new developments in optical sectioning, light microscopic resolution, 3-D imaging and reconstruction, as well as morphometry that are predicated on the confocal microscopic technology and on recent advances in computer software. By making these new advances in light microscopy for structural analyses available to users on the Purdue University campus, the Optical Sectioning and 3-D Reconstruction Laboratory will also stimulate the development of new research opportunities.

vagus nerve; gastrointestinal function; neurotransmitter metabolism; motor neurons; neurons; ganglions; neurochemistry; neuroanatomy; laboratory rat;

The proposed project continues a programmatic investigation of the neural mechanisms underlying the control of feeding and body weight regulation. The long-range goal of the project is a comprehensive functional neuroanatomy of homeostasis. For the immediate future, this goal takes the form of a series of structure-function analyses of the vagus verve. Extensive behavioral and physiological evidence has recently established that the vagus, the Xth cranial nerve, is importantly involved in food intake and body energy regulation. Currently, however, continued progress in analyzing the vagal mechanisms implicated in ingestive behavior and physiology is seriously hampered by a lack of information about the organization of the vagal brainstem nuclei, the distribution of the vagal fibers in the digestive tract and abdominal organs, and the architecture of the forebrain and brainstem mechanisms controlling the different vagal outflows coordinating digestion and energy handling. Hence, the immediate goal of the proposal is the completion of a series of promising analyses, begun during the last period, that are more fully characterizing the topographic and other organizational principles of the last three neuronal relays in the parasympathetic outflow (i.e. the postganglionics, preganglionics, and afferents to the preganglionics) controlling the visceral organs of digestion and metabolism. Two major sets of experiments designed to provide a foundation for later, more focused functional analyses are proposed: The first set will generate separate inventories of the postganglionic targets, the distribution patterns, and the types of terminal profiles for the different pools of motor neurons in the dorsal motor nucleus of the vagus. Additionally, the distributions of the vagal afferents in the abdominal organs will be mapped simultaneously. The second set will begin to delineate the different CNS afferent projections to, and the local circuitry controlling, the identified pools of motor neurons within the dorsal motor nucleus of the vagus. Both sets of experiments will employ highly selective vagotomies, double and triple labeling with neural tracers, and high resolution quantitative microscopy strategies, as well as physiological analyses. The program outlined will extend our understanding of major neural mechanisms implicated in metabolic and digestive diseases including obesity, diabetes, anorexia, disorders of swallowing, vagal dysfunction, peptic ulcers, and eating disorders.

The proposed project continues a programmatic investigation of the neural mechanisms of feeding and body weight regulation. The long- range goal of the project is a comprehensive functional neuroanatomy of energy homeostasis. For the immediate future, the proposed experiments concentrate on a series of structure-function analyses of the vagus nerve. Extensive evidence has established that the vagus, the Xth cranial nerve, is critically involved in controlling ingestion and body energy regulation. Presently, however, continued progress in analyzing vagal mechanisms involved in ingestive behavior and physiology is seriously limited by a lack of fundamental structural information about the vagal projections to the gastrointestinal tract. Hence, the immediate goal of the project is the completion of a series of promising analyses that are characterizing the morphological types, regional topographies and functions of vagal projections linking the brain and GI tract. The first aim is to complete a series of experiments on vagal afferent, or sensory, endings in the alimentary canal. This aim includes characterizing structurally and then classifying afferent terminals in the mucosa and submucosa of the GI tract. It also includes developing topographic maps of the concentrations of these chemo-, osmo-, thermo- and mechano- receptors throughout the GI tract. The second aim is to characterize the patterns and timetables of regeneration of each of the different types of vagal projections to the GI tract. These experiments will also correlate the newly identified patterns of structural plasticity with recovery of function. The third aim is to extend our structure-function analyses by screening mutant mice with selective afferent vagal ablations for potential alterations in feeding behavior. Inventories of the different vagal endings, surveys of their terminal distributions, and characterizations of the plasticity in these vagal projections produced by damage or mutation will yield information needed to design, guide and interpret both experimental and surgical manipulations of the autonomic nervous system and GI tract. The program outlined also will extend our understanding of major neural mechanisms implicated in metabolic and digestive diseases including obesity, eating disorders, anorexia, disorders of swallowing, diabetes, vagal dysfunction, autonomic neuropathies, visceral pain, irritable bowel syndrome, and peptic ulcers.

To consolidate and augment a new graduate training program in Integrative Neuroscience, this proposal requests five years of support. This new program at Purdue University was initiated three years ago. It is highly integrative, involving 28 faculty trainers from 10 departments in 5 schools. It provides a solid core of training in the fundamentals of neuroscience as well as more specialized training options, spanning across molecular, cellular and systems levels, in three broad thematic areas. The three themes, each providing training relevant to programs and initiatives in mental health fields, are (1) Molecular Signaling, (2) Development and Plasticity, and (3) Behavioral and Cognitive Neuroscience. Students' work with individualized advisory committees as well as a Graduate Training Committee to assure that they receive strong grounding in fundamentals and principles of integrative neuroscience as well as coherent training in an integrative specialty. The resulting curriculum provides a focus and depth of training often found only in more narrow programs, while also achieving the scope and breadth of the best interdisciplinary programs. Novel features of this program interlink it with the entire national and international neuroscience enterprise. Each semester, in addition to the extensive curriculum offered by Purdue neuroscience faculty, a special Integrative Neuroscience course staffed by eminent visiting neuroscientists is offered. Stipends for students to travel to premier training sites to obtain skills in specialized techniques are also provided. The overall result is an exceptional new program. It is integrative both in its content and in the complementarity it provides between Purdue's strengths and the best neuroscience resources available nationally and internationally. Students trained in the interdisciplinary Purdue Integrative Neuroscience program will make substantial contributions to mental health research and its applications in the next century.

DESCRIPTION (provided by applicant): In the elderly, gastrointestinal (GI) disorders are common, often either complicate or are complicated by other diseases, and can be debilitating. Research over the last decade has established that aging-related GI disorders are correlated with dramatic losses (~40 - 50%) of neurons in the autonomic, or enteric, plexuses of the digestive tract. The past research is very limited, however, and has been focused almost exclusively on establishing that losses occur. Too little is presently known to specify underlying mechanisms, to establish causal relationships, or to formulate rational therapeutic interventions. More specifically, too little information is available on which GI regions are compromised, on the neurochemical phenotypes of either the neurons that die or those that survive, and on the temporal patterning of the dissolution. The neuroscience of the aging GI tract has largely focused on the myenteric plexus in a few intestinal regions, while ignoring some organs (e.g., the stomach), some enteric elements (e.g., the submucosal plexus), and the extensive extrinsic motor and sensory innervation (e.g., vagal and sympathetic projections) that links the gut to the central nervous system. The present proposal's long-term objective, which unifies its four specific aims, is to characterize the regional, temporal and neurochemical patterns of neuronal aging in the GI tract. Aim 1 will specify the neurochemical phenotypes of enteric neurons throughout the gut that undergo age-related neurodegeneration. The evolution of such losses over the lifespan will also be delineated. Aim 2 will analyze age-related axonopathies of the extrinsic innervation of the GI tract and determine the temporal patterns by which those pathologies develop. Aim 3 will correlate aging-associated changes in GI motility and nutrient handling with the different specific neuropathies characterized in Aims 1 and 2. Aim 4 will begin to evaluate two particularly promising hypotheses identifying cellular mechanisms underlying the neuronal losses and axonopathies of aging. The four aims will be addressed using a battery of immunohistochemical protocols, anterograde tracing techniques, and functional assays of gut motility patterns employing recently developed spatio-temporal mapping algorithms. These analyses will be done primarily with animal models of aging established by the National Institute on Aging. Taken together, the proposed observations on the aging the GI tract will specify the vulnerable regions, neuronal phenotypes, timepoints, and cellular processes to which therapeutic interventions should be targeted. PUBLIC HEALTH RELEVANCE The proposed understanding of the spatial, temporal and neurochemical patterns of neural losses in the GI tract with aging will provide a much-needed foundation for rational strategies for clinical interventions or management of some of the homeostatic and digestive disorders that affect appetite, ingestion, digestion, and motility, as well ultimately, nutrition and health, in the elderly.

Project II: Gastrointestinal nutrient signals The present obesity epidemic highlights the fact that many individuals are unable to limit adequately their intakes of calories when exposed to modern Western diets. Given the urgency of this health problem, it is problematic that so little is known about gastrointestinal (Gl) nutrient sensing that might be augmented to help limit ingestion. The long-term objective of the present proposal is to characterize and analyze more fully such post-oral or post-ingestive sensing abilities of the Gl tract. Traditionally, nutritional neuroscience has had few experimental strategies for analyzing nutrient detection in the gut, and some of those techniques have not readily linked putative signals to the control of ingestion. To help address these limitations, we have recently introduced a new testing paradigm that assesses whether a particular macronutrient or defined stimulus property of food in the Gl tract can be used to control feeding decisions. This "intestinal taste aversion paradigm" pairs a Gl infusion of a novel nutrient with subsequent malaise to train animals. After conditioning, the animals are then given oral intake tests with the nutrient. This protocol provides a behavioral assay for determining whether an animal can detect, and discriminate between, particular macronutrients in its Gl tract. Such visceral sensory psychophysical information can indicate which features of nutrients are salient and effective in short-term control of ingestion and which features are poorly detected or perhaps go undetected. The present proposal seeks to characterize the neural mechanisms of this intestinal taste aversion and to use the procedure to assess the nutrient-detecting proficiencies of the gut: Specific Aim 1 is to determine where in the Gl tract or organs of metabolism, and by what pathways, different nutrient stimuli that influence intake are detected. Specific Aim 2 is to determine the most effective stimulus and protocol parameters for the paradigm in order to optimize such nutrient feedback and characterize its mechanisms. Specific Aim 3 is to use additional stimuli and generalization tests to determine what discriminations can be made about gastrointestinal stimuli and how broadly or narrowly tuned the post-ingestive nutrient detectors are. Specific Aim 4 is to assess the relationship between learning about post-ingestive nutrient stimuli and learning about nutrient cues detected by mouth. The proposed research program has implications for understanding and treating a variety of eating and metabolic disorders including anorexia, bulimia, and food allergies as well as, of course, obesity. Dr. Rowley (PI) and Dr. Davidson will direct this research in collaboration with Dr. Robert Phillips from the Department of Psychological Sciences at Purdue University.

Address; Afferent Pathways; Alimentary Canal; Animal Testing; Animals; Body Weight; Buccal Cavity; Catheters; Cavitas Oris; Cell Communication and Signaling; Cell Signaling; Chronic; Condition; Consumption; Cues; Diet; Digestion; Digestive Tract; Drugs; Eating; Elements; Emetic Agents; Emetic Drugs; Emetics; Epidemic; Feedback; Food; Food Intake; GI Tract; Gastrointestinal Tract; Gastrointestinal tract structure; Gustation; Head and Neck, Buccal Cavity; Hybrids; Individual; Infusion; Infusion procedures; Ingestion; Intake; Intestinal; Intestines; Intracellular Communication and Signaling; Learning; Malaise; Medication; Mouth; Nature; Nausea; Nutrient; Obesity; Oral cavity; Organ; Pharmaceutic Preparations; Pharmaceutical Preparations; Phase; Preparation; Procedures; Protocol; Protocols documentation; Psychophysic; Psychophysics; Reporting; Rest; Sampling; Satiation; Satiations; Science; Sensory; Series; Signal Transduction; Signal Transduction Systems; Signaling; Site; Societies; Stimulus; Stomach; Taste; Taste Perception; Testing; Training; Visceral Afferents; Wrestling; adiposity; alimentary tract; biological signal transduction; bowel; corpulence; corpulency; corpulentia; design; designing; detection of nutrient; digestive canal; drug/agent; experiment; experimental research; experimental study; gastric; gastrointestinal; novel; nutrient sensing; obese; obese people; obese person; obese population; perception of nutrients; research study; satiety

Objective The overarching objective of the Analytical Core is to facilitate and implement the synergies among the three collaborative and complementary projects that constitute the Program Project Grant (PPG). The Analytical Core is operated so as to achieve three specific aims. The first specific aim is to facilitate additional synergies among the PPG projects through the standardization of common nutrient solutions, stimuli, assay measurements and other variables that will increase extrapolations among the different projects. The second specific aim is to promote synergies among the grant projects by identifying and reporting commonalities shared among the projects in terms of techniques. The third specific aim is to facilitate and enhance the work of the individual projects in the program project, in some cases by offering economies of operation and in other cases by providing extensive services and formulations, assays, access to equipment, or measurements that might not be available to each laboratory, individually.

Project Summary Two series of complementary experiments, conducted by our exceptionally qualified team of 11 investigators from eight state-of-the-art laboratories at four institutions, will close critical gaps in the current characterization of the autonomic connectome controlling stomach function. These proposed synergistic anatomical and functional investigations will form the needed foundation for neuromodulation protocols that can correct shortcomings in past, first-generation bioelectronic attempts to ameliorate and monitor gastric disorders. Building on recent advances in mapping of vagal circuits, many reported by our research team, SA 1 will finish inventories of the efferent and afferent terminal phenotypes, analyze their collateral specializations, establish their regional distributions, and identify chemical taxonomies of their target tissues. We will also compare the neural circuitry of the human (and pig, an ideal large animal preclinical proof-of-principle model) stomach with that of the rat model to facilitate future translational extrapolations. The analyses of SA 1 will use a suite of high-definition neural tracing, immunohistochemical and molecular protocols, along with advanced imaging and morphometric techniques, that our team has adapted to autonomic circuits. SA 1 will focus on those elements in gastric neural network that may be most relevant to informing SA 2 and identify additional localized sites in the stomach wall where focal stimulation will have strong therapeutic potential. In functional assessments, SA 2 will identify optimal locations for both highly selective vagal stimulation (VNS) and precise surgical placement between the exit of the vagus from the brainstem and the target sites of the axons within the stomach wall and will determine the best stimulation protocols for augmenting gastric physiology. SA 2 will use state-of-the-art closed-loop stimulator technologies, algorithms, and electrodes (previously designed and robustly proven by investigators on our research team) and assess the reliability, validity, and stability of the VNS by employing a battery (? 6) of acute, short-term, and long-term non-invasive endpoints. The team will use data archiving and resource sharing platforms that are universally accessible to all research and medical communities.