Thomas E. Finger - US grants

Taste buds are situated at the junction of the external world and the alimentary canal. The sense of taste has been shown to play roles both in food selection and ingestion. This functional division corresponds to an anatomical division in terms of innervation of different groups of taste buds. Taste buds implicated in ingestive functions are situated closest to the esophagus and are innervated by the vagus nerve; taste buds involved in food selection are located closest to the external world and are innervated by the facial nerve. Our work has shown that taste inputs from these two gustatory nerves are procesed in different locations within the central nervous system. The disposition of glossopharyngeal nerve gustatory inputs is unknown and will be examined in the coming grant period. The proposed studies will compare and contrast the central organization of the different gustatory channels. Connections of the primary and secondary gustatory nuclei will be examined by means of horseradish peroxidase tracing techniques. The mode of termination of primary afferent fibers of the different gustatory nerves will be compared at light and electron microscopic levels. The synaptic organization of primary gustatory nuclei will be determined by means of combined degeneration and retrograde transport techniques. In addition, those areas of the CNS involved in regulation of digestion will be determined in order to delineate any areas of overlap between gustatory and digestive centers.

The sense of taste is important both for food selection and digestion. The gustatory system can be divided into two functionally and anatomically distinct channels, a facial nerve channel and a vagal nerve channel. These two gustatory channels each maintain distinct sensory regions in the medulla and higher levels of the neuraxis. A comparison will be made of the central nervous system structures involved in each of these gustatory channels. Connections of the gustatory nuclei will be examined by means of autoradiographic, degeneration and enzyme (horseradish peroxidase) tracing techniques. Differences and similarities in neurotransmitters and neuropeptides involved in the gustatory nuclei will be sought by means of immunocytochemical and histochemical methods. The morphology and synaptic interrelationships of the various gustatory nuclei will be examined at the light and electron-microscopic levels. The possibility of growth in the vagal lobe will be examined by tritiated thymidine techniques.

The aim of this multidisciplinary program project is to gain a better understanding of the cellular mechanisms and interrelationships underlying chemosensory transduction and transmission of information in both human and animal model systems. Research under the auspices of the Rocky Mountain Taste & Smell Center will continue to concentrate on a cellular level analysis of structure/function relationships in gustatory and olfactory receptors. The Center comprises five laboratories in the greater Denver metropolitan area. The ionic and second messenger systems involved in transduction in taste buds will be studied with modern patch-clamp methodology. In addition, the relationship between chemospecificity and morphology of taste receptor cells will be determined. Other studies will examine the interactions between receptor cells within single taste buds. The possibility of electrical as well as chemical interactions between cells will be examined. The relationship between taste cell morphology and lineage will be studied in chimeric mice. These experiments will test whether the different morphological types of cells within taste buds represent different cell lines or different morphological manifestations of a single cell line within each taste bud. In addition, the development of taste buds will be studied to determine the sequence of origin of different cell types, synapses and opening of the taste pore. Structure/function correlation studies will examine whether the changes in chemospecificity accompanying development are mirrored by discrete anatomical changes in the receptor cell-nerve fiber interrelationships. Clinical and animal models will be utilized in studies of the olfactory epithelium. Preliminary results demonstrate unique crystalline deposits in the olfactory epithelium of presumed Alzheimer's disease patients. Examination of the patient biopsies with an KEVEX system will permit determination of the exact atomic nature of the crystals, as well as their prevalence in the biopsy material. An animal model of Alzheimer's, based on disruption of mitochondrial function, will be studied for similar histopathologic changes in the olfactory epithelium. Finally, the role of the nasal microvillar cell will be investigated further in order to test the possibility that these cells play a role in nasal chemoreceptor systems.

The gustatory system is situated at the entrance to the alimentary canal and serves a variety of reflex functions including oropharyngeal control, e.g. swallowing and gagging, as well as regulation of the gastrointestinal (GI) tract. The proposed studies are designed to test the hypothesis that two or more gustatory submodalities exist, each being involved in different reflex systems, and therefore each maintaining different connections within the brainstem. The hypoethesis will be tested by comparing, by anatomical and physiological means, the reflex connections of the primary gustatory nuclei devoted to facial nerve taste, glossopharyngeal nerve taste, or vagal nerve taste. Such studies on the interrelationship of the gustatory and general visceral systems are not feasible in mammals since the primary gustatory and primary general visceral nuclei are not clearly segregated within the medulla. In highly gustatory fish such as catfish and goldfish, however, these studies are practical. In the first phase of this study, anatomical tracing methods will be used to delineate the medullary general visceral sensory and motor nuclei involved in innervation of the GI tract. The three different primary gustatory nuclei, facial lobe, glossopharyngeal lobe, and vagal lobe, then will be injected with anterograde tracers to determine whether direct connections exist from any of the primary gustatory nuclei onto any of the GI tract sensory or motor nuclei found in the first experiments. Extracellular electrophysiological methods will be used to characterize the nature of any gustatory information reaching these general viscerl control nuclei. In the second phase of the project, the previously demonstrated reflex system connecting vagal taste centers to the nuc. ambiguus will be investigated by anatomical and electrophysiological means, including both in vivo and in vitro methods. The final two phases of this study will investigate whether hypothalamic visceromotor control centers are involved in gustatory regulation of general visceral activity. Two specific questions will be addressed: first, whether the gustatory submodalities remain separate at hypothalamic levels, and second, whether gustatory afference reaches areas of the hypothalamus that project onto the visceromotor nuclei defined in the first phase of th experiment. At their completion, these studies should delineate the pathways involved in gustatory modulation of general visceromotor activity.

Colorado houses several investigatory teams engaged in research on basic mechanisms and clinical problems relating to the chemical senses of taste and smell. This proposal seeks funds to develop the informational and outreach capabilities required for a NIH multipurpose Research and Training Center (RTC) in Chemosensory disorders. In addition, funds are sought to expand the research capabilities of investigators into the basic biological mechanisms underlying the senses of taste and smell. Three pilot research projects are proposed as well as requisite outreach surveys and curriculum development. The first pilot research project will test the adequacy of a small animal model for irradiation-induced dysgeusia. To date, no adequate model system has been developed incorporating both behavioral and anatomical measures of radiation-induced changes. The second pilot project entails attempts to develop a defined culture system in which to examine the trophic interactions between sensory nerves and taste buds. The third pilot project involves application of new technology (in vivo electrochemistry) to measure diffusion of odorants in the olfactory mucus. Definition of this process is essential to our complete understanding of how odorants reach the receptor cilia. The final component of this exploratory proposal centers on continuing education. Although a substantial outreach program exists at the Univ. Colorado, there is no focus on chemosensory disorders. During the proposed pilot program, a needs assessment will be carried out and a suitable curriculum developed relating to chemosensory dysfunctions. At the completion of the exploratory funding period, the Center will be well positioned for a full RTC including strong programs in research, research training, information dissemination and continuing education.

Taste buds and olfactory epithelium each consist of several morphologically-identifiable cell types. The proposed experiments will examine whether these different cell types arise from different progenitor cells and, for taste buds, whether different morphological types are related to the immunochemically distinct cells in a taste bud. In order to examine lineage relationships within chemosensory epithelia, chimeric mice will be prepared in which embryos from two different mouse strains are fused early in development. The resulting chimeric mouse consists of tissues exhibiting a mosaic of cells derived from one or the other strain. The pattern of mosaicism in relationship to the different morphological types of cells identifiable in the chemosensory epithelia will permit determination of whether the different cell types originate from a common precursor or from different precursors. For example, the patterns in the chimeric mice will permit determination as to whether dark and light cells within a taste bud arise from different progenitors or whether these taste cells represent endpoints of a single lineage. Further, these experiments may reveal whether taste buds are clonal populations of receptor cells derived from single progenitors. If so, the number of such taste bud progenitor cells can be determined. Similarly, chimeric mice will be used to study the origins of the various cell types in the olfactory epithelium: receptors, supporting cells and microvillar cells. Whether these three cell types originate from a common precursor should be determinable. Other studies in this proposal will compare cytochemically different cells within a taste bud to determine whether the cytochemical differences reported are related to cell morphology or to functional status. The focus of these investigations will be taste cells expressing robust NCAM immunoreactivity. Such cells do not fit well into a single morphological type (dark, intermediate, light) and the presence of NCAM may reflect the synaptic status of the cell. Reconstructions of serial sections taken through the NCAM-immunoreactive cells will determine whether these cells contact nerve fibers, and if so, whether they form synaptic contacts.

DESCRIPTION: (ADAPTED FROM THE APPLICANT'S ABSTRACT): The sense of taste plays a pivotal role in the selection of potential food items or rejection of potential toxins. Thus appetitive and aversive stimuli must be processed in different ways or in separate neural networks in order to produce different responses. The proposed experiments are designed to delineate the neural networks and transmitters used in dealing with appetitive and aversive stimuli. The primary sensory nucleus for taste in mammals, the nucleus of the solitary tract, is difficult to study because of its compact, complex nature and relatively undifferentiated organization. Accordingly, the proposed work centers on the vagal lobe, a distinctly laminated and highly organized primary gustatory nucleus in a non-mammalian vertebrate. The vagal lobe is organized incortex-like fashion complete with discrete layers,and with columns of incoming afferents. The vagal lobe not only carries out the initial sensory processing of gustatory inputs, but also contains motoneurons equivalent to the nucleus ambiguus that drive the gustatory-related feeding behaviors. Initial experiments will test the animal's behavioral reaction to various amino acids or quinine in order to establish thresholds and classes of stimuli. Then, detailed electrophysiological studies will be carried out in order to determine whether stimulus quality is related to either layer or functional column within the lobe. Output systems of the lobe will be studied by use of intracellular recording and dye-filling of different motoneuron systems. The neurotransmitters and receptors utilized by the primary gustatory afferents and interneuron systems in the lobe will be studied by means of in vitro electrophysiology, immunocytochemistry, in vitro physiology and pharmacology, and ligand binding methods.In particular, the role of acetylcholine, excitatory amino acids and ATP will be tested. Finally, the possibility that cholecystokinin (CCK), a peptide implicated in regulation of feeding behavior and present in the primary gustatory nucleus, plays a role in modulation of gustatory responses will be studied by application of the peptide in the in vitro recording paradigm. Collectively, these studies will reveal the basic circuitry and neurotransmitters involved in gustatory reflex systems controlling essential physiological functions such as swallowing and choking.

Taste determines palatability and ultimate disposition of potential foodstuffs in the oral cavity. The overall goal of this project is to determine the circuitry and neurotransmitters in brainstem nuclei that process taste information. The primary sensory nucleus for taste in mammals, the nucleus of the solitary tract (NTS), is difficult to study because of its compact, complex nature and diffuse organization. Accordingly, the proposed experiments exploit the distinct laminar pattern and large size of the NTS-equivalent in a non-mammalian vertebrate. This NTS-equivalent, the vagal lobe, is organized in cortex-like fashion providing an anatomical separation of primary gustatory afferents from interneuron systems. Our past work has shown that the vagal lobe is amenable to anatomical and physiological studies examining the role of neurotransmitters and circuitry within brainstem gustatory nuclei. The proposed series of experiments tests whether excitatory amino acids meet the criteria for being neurotransmitters at three levels of the gustatory neuraxis: I) primary afferent terminals, ii) reflex systems to vagal motor neurons, and iii) second-order projections to the pontine secondary gustatory nucleus (pontine taste area). First, immunocytochemistry and high-pressure liquid chromatography will be used to test for the presence and potassium-evoked release of excitatory amino acid neurotransmitters. Second, the identity and function of excitatory amino acid receptors in these areas will be studies with radioligand binding and in vitro electrophysiology of vagal lobe slices. Third, radiolabeled analogs will be used to test for the presence of high-affinity uptake systems commonly associated with excitatory amino acid neurotransmitters. A second group of experiments will test whether previously identified peptidergic and serotonergic systems impacting on the vagal lobe can modulate excitatory amino acid neurotransmission with the brainstem gustatory nuclei. The anatomical relationship between primary gustatory afferent terminals and peptidergic/serotonergic terminals will be examined anatomically to test for potential presynaptic contacts between these systems. Finally, the effects of the neuropeptides and serotonin on release and transmission will be studied by means of HPLC and in vitro electrophysiology. The proposed studies will resolve existing controversies regarding the role of excitatory amino acids in gustatory neurotransmission and processing.

stem cells; taste buds; developmental neurobiology; mammalian embryology; embryo /fetus tissue transplantation; epithelium; neurotrophic factors; cell cell interaction; mesenchyme; gene mutation; nervous system transplantation; cell transplantation; animal genetic material tag; embryo /fetus; laboratory rat; laboratory mouse;

Recent advances in imaging and histological technologies have opened the way to visualization of functional and molecular features of tissues. Such techniques are especially apropos for studies on chemosensory epithelia, since these tissues contain a variety of cell types with differing functional roles and receptor specificity. The proposed Core will provide histological, imaging and analysis capabilities to Core investigators, thereby enhancing the research capabilities of all involved. Further the core will serve as a focus for interactions and development of shared analytical tools. Three specific aims are proposed. Molecular and Cytochemical Histological Services Multi-photon imaging Visualization and Quantitative Analysis of 3-Dimensional Anatomical Data Centralization of these services permits optimization of usage and obviates the redundancies attendant to establishing each technical capability in each laboratory. Further, the Core facility enhances interactions among the investigators leading to recognition of common problems in analysis of 3-D data sets. The establishment of Core facilities for these functions and interactions with established chemosensory researchers facilitates entree of three new investigators into the study of the chemical senses.

[unreadable] Description (provided by applicant): The nasal cavity in mammals houses distinct chemosensory epithelia including the main olfactory epithelium, the vomeronasal epithelium and the trigeminally innervated respiratory epithelium (so-called "non- sensory" epithelium) (Finger et al., 2000). Each of these receptive epithelia is implicated in detection of diverse compounds and evokes different behaviors. Traditionally, the vomeronasal epithelium was thought to detect pheromones - chemicals released by a conspecific that elicit a physiological or behavioral response in the recipient - while the main olfactory epithelium was thought to mediate conscious perception of general odorants. In addition, the trigeminal system is thought to detect potentially noxious substances, which can elicit aversive responses such as a decrease in respiratory rate, sneezing or coughing. However, recent experiments - including those published by us in the current period of support - indicate that the traditional view on the role of these chemosensory systems needs to be revised because the main olfactory epithelium also appears to be involved in detecting pheromones and other semiochemicals - odors involved in animal communication (Baxi et al., 2006; Buck, 2005; Lin et al., 2007; Lin et al., 2004). This novel aspect of chemoreception by the main olfactory system will be studied in this proposal. We will focus our proposal on our finding that the transient receptor channel M5 (TRPM5), an effector in the phospholipase C (PLC) pathway, is expressed in a subset of olfactory sensory neurons in the main olfactory epithelium whose axons project to semiochemical-responsive glomeruli (Lin et al., 2007). We propose three specific aims: Aim 1. Test whether the PLC pathway participates in olfactory transduction by opening the TRPM5 channel in response to the increase in calcium elicited by odors. Aim 2. Test the hypothesis that sensory input to the nose during the postnatal period affects TRPM5 expression in OSNs Aim 3. Test whether TRPM5 OSNs transmit information about semiochemicals to restricted areas of the MOB which project in turn to the medial amygdala - an area associated with reproductive and defensive behaviors. In humans, disorders of the sense of smell are encountered in diseases such as Alzheimer's (Doty, 1991;Rawson, 2000), bipolar depression (Hahn et al., 2005) and schizophrenia (Turetsky et al., 2003). This grant will study the basic mechanisms of olfactory transduction in olfactory sensory neurons as well as central projections of the olfactory system. This basic science study is performed within the context of the Rocky Mountain Taste and Smell Center (RMTSC), an entity dedicated to basic and clinically-relevant research on olfaction and taste. Drs. Restrepo and Finger are Co-Directors of the RMTSC. Clinically relevant work within the center includes work on schizophrenia, Downs syndrome and brain inflammation. The clinical studies benefit greatly from solid basic science research, and because of this we expect our current proposal to strengthen the clinically relevant work carried out by the RMTSC. Reference List Doty,R.L. (1991). Olfactory dysfunction in neurodegenerative diseases. In Smell and taste in health and disease, T.V. Getchell, R.L. Doty, L.M. Bartoshuk, and J.B. Snow, Jr., eds. (New York: Raven Press), pp. 735-752. Hahn,C.G., Gomez,G., Restrepo,D., Friedman,E., Josiassen,R., Pribitkin,E.A., Lowry,L.D., Gallop,R.J., and Rawson,N.E. (2005). Aberrant intracellular calcium signaling in olfactory neurons from patients with bipolar disorder. Am. J. Psychiatry 162, 616-618. Rawson,N.E. (2000). Human olfaction. In The neurobiology of taste and smell, T.E. Finger, W.L. Silver, and D. Restrepo, eds. (New York: Wiley-Liss), pp. 257-284. Turetsky,B.I., Moberg,P.J., Owzar,K., Johnson,S.C., Doty,R.L., and Gur,R.E. (2003). Physiologic impairment of olfactory stimulus processing in schizophrenia. Biol. Psychiatry 53, 403-411. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The sense of taste plays a pivotal role in regulation of food intake and in determining the palatability of potential foodstuff. The overall goal of the proposed studies is to gain an understanding of the neurotransmitters and circuitry involved in regulation and processing of taste inputs within the primary taste nuclei of the brainstem. The primary taste nucleus in the brainstem of mammals, the nucleus of the solitary tract (NTS) is difficult to study because it contains numerous functional domains packed into a poorly differentiated, small structure. The proposed experiments take advantage of the distinct boundaries and laminated organization of the equivalent structure in a non-mammalian vertebrate. In this NTS equivalent, the vagal lobe, the primary gustatory afferents terminate in discrete layers, making the system amenable to both physiological and anatomical analyses that could not be carried out in other vertebrates. Glutamate, acting via both NMDA and non-NMDA ionotropic glutamate receptors, is the neurotransmitter of the primary gustatory afferents. The proposed experiments focus on early stage regulation of gustatory inputs by glutamate autoreceptors, ionotropic and metabotropic GABA receptors, and neuropeptides. Experiments in the first aim test whether glutamate autoreceptors situated on primary afferent terminals regulate synaptic transmission at the primary afferent terminals. We will utilize calcium imaging of an in vitro slice preparation to study the effects of ionotropic and metabotropic glutamate receptor agonists and antagonists on transmission by primary afferents. In addition, anatomical methods will be used to localize glutamate receptors in relation to the primary afferent terminals. The second aim relies on functional imaging and immunocytochemistry to determine whether GABA acts via presynaptic receptors to modulate primary gustatory inputs. In addition, we will define the GABAergic circuitry of the vagal lobe. The proposed experiments will determine mechanisms involved in regulation of transmission of primary gustatory input to the brainstem.

DESCRIPTION (provided by applicant): Taste buds consist of numerous elongate modified epithelial cells, some of which serve as the transducing elements for taste. Different taste cells are involved in transduction of different taste qualities. Experiments in this R21 proposal are designed to generate and test a transgenic mouse in which under the influence of cre-recombinase, synaptically-connected taste cells will produce the transneuronal tracer, Wheatgerm Agglutinin (WGA). The WGA should cross the synapse into the gustatory nerve fibers thereby labeling ganglion cells and perhaps terminals within the nucleus of the solitary tract. If sufficient WGA is transported, the tracer may also label postsynaptic cells in the nucleus of the solitary tract. The construct to be employed for generation of the mouse is a knock-in whereby the BDNF coding region is flanked by Iox sites and followed by the WGA coding sequence. When acted upon by cre-recombinase, the BDNF coding region is excised and WGA is produced instead. We will use various existing transgenic lines to express crerecombinase in relevant BDNF-expressing populations in cranial ganglia and the brainstem as well as in taste buds. For selective recombination in taste buds, we will utilize an existing K14tamcre line where crerecombinase can function only in keratin 14-expressing cells under the influence of tamoxifen, thus providing us with good spatial and temporal control. Since basal cells of taste buds express keratin 14, tamoxifen treatment will activate cre-recombinase thereby resulting in production of WGA by taste cells that would have produced BDNF. The population of taste cells that normally express BDNF are synaptically-connected cells that include those specifically implicated in "sour" (H'+)transduction. Thus the WGA-expressing transgenic mice that we propose will specifically mark taste cells and ganglion cells involved in "sour" transduction. These mice also will be informative about the kinetics of protein handling, exocytosis and uptake by various cell types within the taste bud.

DESCRIPTION (provided by applicant): Taste buds consist of numerous elongate modified epithelial cells, some of which serve as the transducing elements for taste. Different taste cells are involved in transduction of different taste qualities. Experiments in this R21 proposal are designed to generate and test a transgenic mouse in which under the influence of cre-recombinase, synaptically-connected taste cells will produce the transneuronal tracer, Wheatgerm Agglutinin (WGA). The WGA should cross the synapse into the gustatory nerve fibers thereby labeling ganglion cells and perhaps terminals within the nucleus of the solitary tract. If sufficient WGA is transported, the tracer may also label postsynaptic cells in the nucleus of the solitary tract. The construct to be employed for generation of the mouse is a knock-in whereby the BDNF coding region is flanked by Iox sites and followed by the WGA coding sequence. When acted upon by cre-recombinase, the BDNF coding region is excised and WGA is produced instead. We will use various existing transgenic lines to express crerecombinase in relevant BDNF-expressing populations in cranial ganglia and the brainstem as well as in taste buds. For selective recombination in taste buds, we will utilize an existing K14tamcre line where crerecombinase can function only in keratin 14-expressing cells under the influence of tamoxifen, thus providing us with good spatial and temporal control. Since basal cells of taste buds express keratin 14, tamoxifen treatment will activate cre-recombinase thereby resulting in production of WGA by taste cells that would have produced BDNF. The population of taste cells that normally express BDNF are synaptically-connected cells that include those specifically implicated in "sour" (H'+)transduction. Thus the WGA-expressing transgenic mice that we propose will specifically mark taste cells and ganglion cells involved in "sour" transduction. These mice also will be informative about the kinetics of protein handling, exocytosis and uptake by various cell types within the taste bud.

Taste receptor cells transduce chemical signals into a neural message transmitted to the gustatory nerve fibers that innervate taste buds. This proposal describes experiments designed to test the hypothesis that in response to stimulation, taste cells release ATP which then activates ionotropic purinergic receptors (P2X2 and P2X3) on the gustatory nerve fibers. Previous studies have demonstrated the presence of these two receptors on intragemmal gustatory fibers. Our preliminary data show that gustatory nerves in P2X2/P2X3 double-knockout mice do not respond to any tastants applied to the oral cavity although the nerves give robust responses to touch or thermal stimuli. Similarly, the P2X2/3 KO mice do not respond behaviorally to many tastants including sweeteners, and many bitter substances although they do respond aversively to citric acid and other bitter substances. This spectrum of responsiveness matches that of the superior laryngeal nerve which innervates non-taste bud chemoreceptors on the larynx. The proposed experiments fall into 3 aims: first to test whether P2X receptors are necessary for transmission of taste information. For these studies we will characterize the system behaviorally and electrophysiologically to test the extent of function of the lingual gustatory system. We will also test whether laryngeal chemoreception accounts for remaining behavioral capabilities of these KO mice. Second, we will utilize anatomical and physiological measures to test whether taste buds in the knockout mice are normal both in terms of structure and function. We will assess structure and function of taste buds in P2X2/3 KO mice to test whether the receptor cells are normal and that no overt changes in the receptor epithelium may underlie the loss of taste function in the P2X2/3 KO mice. In the final aim, we will test whether taste buds in both wildtype and knockout mice release ATP upon stimulation. These functional studies will be carried out in vitro both by using the luciferin- luciferase assay and by ATP-sensing biological probes (so-called "sniffer-cells"). In addition, we will utilize quinacrine loading to assess vesicular uptake and release of ATP by taste cells. Taken together, the proposed studies will test the role of ATP and ionotropic purinergic receptors in transmission of taste information.

Description (provided by applicant): The nasal respiratory epithelium contains thousands of specialized chemoreceptor cells (solitary chemosensory cells) that form functional contacts with the trigeminal nerve. Activation of the trigeminal nerve either directly by irritants or through the agency of these cells evokes protective airway reflexes such as sneezing, coughing or apnea. The proposed experiments investigate the role of these solitary chemosensory cells in the detection of quorum sensing molecules secreted by pathogenic bacteria as they transition from being benign to the virulent state. Our preliminary data show that the cells and the trigeminal system at large can respond to bacterial signaling molecules. The experiments in this proposal will examine the chemical specificity of the response, test the transduction cascade and possible role of T2R (bitter taste) receptor molecules, and finally examine the effects on the surrounding epithelium and the trigeminal sensory nerve fibers of activation of the chemosensory cells. In order to assess the effectiveness of various bacterial signaling molecules, we will use two bioassay systems: i) respiratory reflexes evoked by application of the compound to the nasal epithelium in a semi-intact preparation, and ii) Ca ++ -imaging of chemosensory cells isolated from the epithelium of transgenic mice in which GFP marks the relevant cell population. We will use the same preparations to assess the potential role of T2R receptors and the associated PLC-signal cascade. Specific blockers of PLC- signalling should disrupt transduction and eliminate the Ca ++ signal if the T2R/PLC pathway is necessary. Similarly, respiratory depression should be lessened in both TRPM5 and gustducin-knockout animals if these elements are crucial for the transduction of bacterial signals. Finally, we will assess whether activation of the chemosensory cells secondarily causes changes in the surrounding epithelium - either via release of paracrine mediators (e.g. ATP, acetyl choline) or through the agency of activation of the peptidergic nerve fibers that innervate the epithelium. Taken together, these experiments will determine the mechanisms used by solitary chemosensory cell to detect the bacterial signaling molecules and whether the cells are instrumental in provoking a local tissue and/or immune response to the potential pathogens. PUBLIC HEALTH RELEVANCE: The proposed research will investigate a newly discovered nasal chemosensory system that detects molecules that regulate the virulence of pathogenic bacteria. This research is designed to test the possible role of these sensors in a first line of tissue defense against bacterial nasal and upper respiratory infections.

Administration The administrative structure of the RMTSC is modest (-7% of the total direct costs), and focuses on maximizing interaction among members of the RMTSC and in facilitating efficient provision of sen/ices by the research cores. The Co-Directors of the Center are Dr. Diego Restrepo (P.l.) and Thomas Finger (Co.P.I.). They both have considerable administrative experience. Their responsibility is the overall management and coordination of the Core Center. The two Co-Directors have interacted well during the present period of support. Kate Beatty Administrator of the Department of Cell and Developmental Biology (CDB) and Richard Schwiderski, Grants Specialist of CDB, who have considerable administrative and grants management experience, perform day-to-day administrative procedures. A Management Team consisting of the two Co-Directors and the Directors of the cores meets often (at least once per week) to specifically discuss allocation priorities, usage of cores, future RMTSC meetings, space allocation and budgetary issues. The members of the RMTSC are highly interactive, and this administrative structure has proved to be flexible, yet solid during the present period of support. There are two key regular meetings of the RMTSC members. The biweekly Chemosensory Journal Club serves as a forum for regular interactions among all the members of the RMTSC, while the semi-annual meeting of RMTSC members serves to assess progress and coordinate collaborations among investigators.

Understanding brain pathways and neurotransmitter systems that regulate ingestion is essential to development of pharmacological tools to help patients regulate food intake. Taste is a major factor driving over‐eating so it is important to understand the neurotransmitter systems involved in early transmission of taste information to the brain. The experiments in this proposal will test the proposition that good‐tasting (appetitive) foods, i.e. those rich in sugars and glutamate, activate neurochemically distinct circuits in the brainstem taste relay nuclei than do unpleasant‐tasting (aversive) tastes. The experiments rely on induced expression of the immediate‐early gene, c‐fos, as a marker of taste‐activated neurons. We will compare neuronal populations activated by appetitive substances to those activated by aversive (bitter, sour) ones and how these neuronal populations correlate with specific feeding‐related neuropeptide and transmitter systems of the brainstem taste relay nuclei. In addition, we will study whether taste quality representation in the brainstem is altered in mice with induced dysfunction (genetic knockout) in the detection of particular tastes. Finally, we will test whether neuronal activation in the primary taste nucleus is related to specific connectivity with taste axons contacting taste cells [unreadable]tuned[unreadable] to detect sweet and umami qualities.

Understanding brain pathways and neurotransmitter systems that regulate ingestion is essential to development of pharmacological tools to help patients regulate food intake. Taste is a major factor driving over‐eating so it is important to understand the neurotransmitter systems involved in early transmission of taste information to the brain. The experiments in this proposal will test the proposition that good‐tasting (appetitive) foods, i.e. those rich in sugars and glutamate, activate neurochemically distinct circuits in the brainstem taste relay nuclei than do unpleasant‐tasting (aversive) tastes. The experiments rely on induced expression of the immediate‐early gene, c‐fos, as a marker of taste‐activated neurons. We will compare neuronal populations activated by appetitive substances to those activated by aversive (bitter, sour) ones and how these neuronal populations correlate with specific feeding‐related neuropeptide and transmitter systems of the brainstem taste relay nuclei. In addition, we will study whether taste quality representation in the brainstem is altered in mice with induced dysfunction (genetic knockout) in the detection of particular tastes. Finally, we will test whether neuronal activation in the primary taste nucleus is related to specific connectivity with taste axons contacting taste cells ¿tuned¿ to detect sweet and umami qualities.

DESCRIPTION (provided by applicant): Taste buds are complex multicellular endorgans essential for detection of taste. The cells of a taste bud distinguish between the nutritious and the potentially toxic, and communicate this information to the taste nerves innervating them. Despite recent advances in understanding the molecular signaling underlying taste transduction, a complete understanding of cell--cell interactions and transmission of information to the peripheral nervous system is lacking. We propose to use a newly invented technology, serial blockface scanning EM, to generate a complete 3D picture of taste bud structure. This will permit detailed analysis and determination of several key features including: 1) whether individual nerve fibers make functional contact with more than one cell type in a bud, 2) whether Type I cells fully separate the other cell types or whether there is potential for functional connections between the molecularly different chemosensor elements, and 3) whether atypical mitochondria are unique organlees lying only at points of specialized contact between taste cells (for bitter, sweet and umami) and nerve fibers. This greater understanding of the detailed structure of taste buds will help investigators formulate data-based hypotheses regarding functional interactions between the different cellular elements and a more clear understanding of how taste information is transmitted to the nervous system.

DESCRIPTION (provided by applicant): The presence of acidic substances in the mouth evokes a sensation of sour taste. But oropharyngeal acidification is not only attributable to ingested substances, but may be produced by gastric reflux which evokes strong respiratory and salivary reflexes as well as a sensation of disgust. Acidic substances in the oral cavity not only trigger the taste system but also activate pH-sensitive general mucosal nerve fibers. Previous studies have identified the subset of taste cells necessary for sour transduction, but how these cells transmit the information to the gustatory nerves and thence the brainstem gustatory relay nuclei remains unclear. Acid-responsive mucosal nerve fibers also transmit information about intraoral pH and end within the brainstem in a partially overlapping fashion with the sour taste fibers. Experiments in this proposal utilize behavioral, pharmacological, anatomical and physiological means to examine to what degree the sour taste and non- taste systems contribute to the detection and avoidance of acids. The experiments rely on unique knockout rodent models to dissociate the functions and roles of the two intraoral acid-responsive systems. We will use both pharmacological and genetic tools to study what areas of the brainstem are activated by stimulation of one system in the absence of the other.

? DESCRIPTION (provided by applicant): Taste buds are complex multicellular endorgans essential for detection of taste. The cells of a taste bud distinguish between the nutritious and the potentially toxic, and communicate this information to the taste nerves innervating them. Despite recent advances in understanding the molecular signaling underlying taste transduction, a complete understanding of cell-cell interactions and transmission of information to the peripheral nervous system is lacking. We propose to use a newly invented technology, serial blockface scanning EM, to generate a complete 3D picture of taste bud structure from both mice and humans in order to test whether taste information is transmitted along dedicated labeled lines dedicated to an individual taste quality, or whether the brain receives a more complex signal from the taste buds. The novel EM technology will permit detailed 3D reconstruction of the taste bud connectome allowing for analysis of key features including: 1) whether individual nerve fibers make functional contact with more than one cell type in a bud, and 2) whether the glial-like Type I cells fully separate the other cell types or whether there is potential for functonal interactions between receptor cells encoding the different taste qualities. This greater understanding of the detailed structure of taste buds will help investigators formulate data-based hypotheses regarding functional interactions between the different cellular elements and a more clear understanding of how taste information is transmitted from taste buds to brain. RELEVANCE: Taste buds are complex sensory endorgans crucial for detection of both nutrients and toxins. While many of the molecular mechanisms underlying taste have been elucidated during the last decade, we still do not understand the functional organization and interrelationships between the diverse cells of a taste bud. Investigations in this proposal will rely on new technology, serial blockface EM, to generate detailed 3- dimensional images of taste buds to resolve questions about neural connectivity and cell-cell interactions in transmission of taste information from taste bud to brain.

PROJECT SUMMARY Disruption of the sense of taste is a common feature of COVID-19. The proposed supplement will investigate potential histopathological changes in taste buds and the surrounding epithelium of the tongue in patients who have lost their sense of taste during COVID-19. Biopsies of fungiform papillae will be obtained from a taste center in Germany which shall conduct psychophysical testing prior to sampling 4 fungiform papillae from patients recovering from COVID-19 ? either with or without taste loss. We will analyze these samples molecularly and histologically to test for presence of residual virus and for changes in taste bud number, morphology or cellular composition.