Sue C. Kinnamon - US grants

DESCRIPTION (Adapted from the Investigator's Abstract): Taste is initiated when sapid stimuli interact with receptor sites on the apical membrane of taste receptor cells. This ultimately leads to an increase in intracellular calcium and release of transmitter onto gustatory afferent nerve fibers. The role of the taste cell specific G-protein, gustducin, in coupling bitter stimuli with taste cell response will be examined using a transgenic line of mice in which the gustducin promoter has been linked to the gene for Green Fluorescent Protein (GFP). In these mice, cells expressing gustducin will also express GFP and can be readily identified for functional studies by their fluorescence. A combination of Ca2+-imaging and patch-clamp recording will be used to examine the role of gustducin in bitter taste transduction. Aim 1 will determine if bitter taste responses are limited to gustducin-expressing taste cells, identify which second messenger pathways are involved in this process and assess the specific role of gustducin. Aim 2 will identify which, if any, membrane conductances are activated or modulated by bitter stimuli. Aim 3 will determine if a subset of taste cells is specifically tuned to bitter stimuli and if the same taste cells respond to different types of bitter stimuli. The results of these studies will reveal important insights about the role of gustducin in the transduction and coding of bitter taste.

DESCRIPTION (provided by applicant): Sweet taste transduction begins with the binding of sweet tasting compounds to membrane receptors on taste bus cells, followed by activation of G proteins and change in the levels of 2nd messengers. The 2nd messengers block a K+ channel to depolarize taste cells, increase intracellular Ca2+, and eventually, activate gustatory afferent neurons. Although much has been learned recently about the molecular identities of sweet taste receptors, comparatively little is know about the downstream events linking the receptors to the effector ion channels. Our goal for the proposed experiments is to elucidate the molecular cascade forming this link. Previous studies on sweet taste transduction have examined lingual taste buds, which are relatively insensitive to sweet in rodents. Our proposal focuses instead on that buds of the soft palate, which, in rodents is most sweet-sensitive taste field. Cyclic AMP cGMP and IP3 have been implicated as 2nd messengers in sweet transduction, but no single study has examined all three messengers in any one field, nor have the roles of the G proteins and effector enzymes involved in these modulation been assessed. We Present evidence that sweet transduction in the soft palate is mediated in part by the G protein, Galpha-gustducin. We will utilize transgenic mice expressing green fluorescent protein (GFP) in gustducin-lineage cells to select taste cells for electrophysiological recordings. The morphological marker is available in both wild type (Gus-GFP) and gustducin knockout ( gus-null-GFP) mice. Recording form GFP-labeled and unlabeled palatal taste cells and biochemical measurements of 2nd messengers will define the roles of alpha-gustducin in sweet transduction and will reveal whether gustducin-independent mechanisms also exist for sweet transduction. Using a combination of molecular, biochemical, and electrophysiological techniques, we will (1) identiy the G proteins activated by sucrose and synthetic sweeterners. (2) defne the isoforms of adenyly cylcase present in sweet-sensitive taste cells, and (3) determine the mechanisms by which second messengers modulate the sweet-sensitive conductance. Results from these studies will provide important information about downstream signaling mechanisms in sweet taste transduction.

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.

The overall goal of the new Core C is to provide resources, expertise, and training for RMTSC investigators to characterize the chemosensory capabilities and responsiveness of their model rodents. These include phenotypic assessment of transgenic and knockout mice, as well as organisms with experimentally induced taste and olfactory deficits, including localized delivery of pharmacological agents. The need for an assessment core has arisen because the numbers of gene-targeted and transgenic mice produced and/or utilized by established and new RMTSC core investigators has increased dramatically since the last funding period. Further, several new lines are being developed that will be utilized by multiple RMTSC investigators. These model organisms will require functional validation, both electrophysiological and behavioral. Electrophysiology core services include assistance with chorda tympani and glossopharyngeal taste nerve recording, to assess taste function in anterior and posterior lingual fields, and EOG recording, to assess function of the olfactory epithelium and olfactory nerve conduction. Behavioral assessment facilities include training and expertise in the design and execution of behavioral testing for both taste and olfactory function. These services include assistance with design and analysis of brief access taste tests using the Davis Rig llckometer, long-term 2-bottle preference tests, and olfactory function using the olfactometer. In addition to providing services for established investigators. Core C will aid new investigators who do not have the equipment or expertise to perform these assessments on their own. Further, having central core facilities will enhance collaborations among RMTSC investigators, and bring new investigators into the chemosensory field. It is not the intention of this core to perform all these studies for the participating laboratories, but rather to facilitate the capabilities of the participating laboratories to gain the requisite expertise.

DESCRIPTION (provided by applicant): Taste buds are the transducing elements of gustatory sensation. As such, they represent the first step in the process by which nutritious substances, such as Na+ salts, sugars, and amino acids can be detected and distinguished from harmful substances, such as acids and toxic bitter compounds. Understanding the steps in this process may lead to therapeutic interventions that can be used to modulate food intake, a critical factor in controlling obesity. Each taste bud comprises three types of elongate taste cells and a population of basal cells. Understanding the functional role of each cell type is fundamental to understanding how each type of stimulus is detected and how this information is transmitted to the nervous system. Of the three types of fusiform cells, the Type II cells, which express the receptors and signaling effectors for bitter, sweet, and umami transduction, are the best understood. They release ATP to activate purinergic receptors on afferent nerve fibers. Type III, or presynaptic cells, detect sour stimuli and release serotonin and noradrenalin. Type I, or glial-like cells are the most abundant cells in the taste bud but also the least understood. Similar to glial cells in the nervous system, their membranes closely envelope other taste cells and they express NTPDase2, an ectoATPase that degrades ATP that is released from Type II cells. Recent data suggest that Type I cells may have additional functions, including transduction of Na+ salts and modulation of Type II cells. Unlike Type II and Type III cells, there are no fluorescent reporters for identification of Type I taste cells, making identification and functional characterization difficult. The proposed studies will utilize existing reagents and technology to develop a gene-targeted mouse that will express a nuclear-targeted fluorescent reporter (GFP) and Cre recombinase from the NTPDase2 promoter. Nuclear localization of GFP will insure that Type I cells can be distinguished from the other cell types, both within the bud and after isolation. Moreover, Cre recombinase will allow selective deletion of sequence from Type I taste cells, when these mice are crossed with mice carrying an appropriate floxed allele. In the first aim, we will validate the expression of the targeted allele, by crossing the mce to a commercially available Cre reporter line, Rosa26-tdTomato, which will express a red fluorescent reporter upon Cre-mediated excision of sequence flanked by loxp sites upstream of the reporter. In the second aim, we will utilize the mice to test the hypothesis that Type I cells are necessary for amiloride-sensitive salt taste. Whole cell recording will test whether Type I cells express functional amiloride- sensitive Na+ currents. Further, we will develop the methodology to specifically ablate type I taste cells, using the diphtheria toxin receptor targeted to Type I taste cells. In summary, these experiments will allow us define the Type I cell population and provide a new tool to dissect the functions of this most common taste cell type.

DESCRIPTION (provided by applicant): While substantial progress has been made in identifying the receptors and downstream signaling mechanisms involved in taste transduction, much less is known about how taste cells transmit this information to the nervous system. Recently we identified ATP as a key transmitter linking taste cells to activation of gustatory afferent fibers. Using mice that lack the purinergic receptors P2X2 and P2X3, we showed that these mice lack gustatory responses to all taste stimuli. Further experiments by our labs and others have shown that the taste cells that express bitter, sweet, and umami taste receptors release ATP via non-vesicular mechanisms, likely hemichannels composed of Pannexin1. Further, the ATP that is released is broken down to ADP by the ecto- nucleotidase NTPDase2, expressed on the membranes of the glial-like support cells in the taste bud. However, several questions remain about the role of ATP as a transmitter in taste buds. First, is Pannexin1 required for ATP release and full activation of gustatory afferents? This has not been tested in vivo. Further, why are sour and salty responses absent in the P2X2/3 double knockout mice when the only taste cells known to release ATP are the cells that respond to bitter, sweet, and umami stimuli? Finally, what is the role of the ecto-ATPase in the perdurance of the ATP released from taste buds and how does this impact gustatory function? In this new grant proposal we will use existing knockout mice to genetically eliminate key elements of purinergic signaling in taste buds. We will use an integrated systems neuroscience approach-- from cellular assays of ATP release to gustatory nerve recording and behavior-- to answer the following questions: (1) Is Pannexin1 required for ATP release and activation of gustatory nerve fibers? We will use both a global and a conditional knockout of Pannexin1 to address whether this channel mediates ATP release in taste cells, and whether it is necessary and sufficient for full activation of gustatory afferent fibers and taste guided behavior. (2) What is the role of NTPDase2 in the perdurance of ATP release? We will utilize NTPDase2 knockout mice to determine how NTPDase2 affects the magnitude and perdurance of the ATP that is released and what role this plays in the sensitivity of the afferent nerve fibers to taste stimuli. Results from these experiments will provide important new data about the role of ATP in gustatory function. By understanding how taste cells communicate with afferent nerve fibers, it may be possible to develop pharmaceutical agents that will interfere with this process and modulate taste function. This could have an important impact on health problems that result from disorders of food intake. PUBLIC HEALTH RELEVANCE: The sense of taste evolved to allow discrimination of nutritionally important compounds from toxic substances. However, overconsumption of palatable foods can lead to obesity and Type II diabetes, now a major world heath problem. Conversely, the off-taste of a medicine can be a limiting factor in patient compliance. A better understanding of the mechanisms used by taste cells to detect chemicals and transmit taste information to the nervous system may lead to the development of pharmaceuticals that can control overconsumption and limit the bitter tastes of many drugs.

DESCRIPTION (provided by applicant): Rapid introduction of basic science discoveries into clinical fields requires close collaboration of basic and clinician scientists. Accomplishing this n otolaryngology requires a multidisciplinary approach to better define and treat the many disorders of the head and neck. Residency programs today offer limited research training for otolaryngologists, but few become independent investigators. In contrast, the basic sciences postdoctoral training offers little exposure to the clinical setting, making translational research difficult. The goal of this application is to provide research training in otolaryngology and its related sciences. Support is requested for in-depth training for 1) residents, 2) postdoctoral fellows, 3) predoctoral students and 4) short-term medical students. All trainees will receive an interactive basic research experience with ongoing exposure to and interaction of trainees in the clinical setting through conferences and courses. The pre- and postdoctoral trainees will have a 24-month block of training. Research training for MDs will begin in medical school with students doing short-term (3-month) projects and continue through the residency program with a 2-year block midway through the clinical training. One resident will be admitted each year into this research track. Early introduction and continued research involvement throughout the residency will increase our ability to attract academically oriented faculty members into the field, with backgrounds to become independent investigators. This cross-field exposure will enhance the experience for trainees and promote clinical and basic science interactions as a faculty. A major strength of the program is drawing members of the faculty from a wide variety of departments of the School of Medicine and across the UC campuses involved in otolaryngology related research. This will enhance collaborative efforts in related fields of hearing, balance, smell, taste, speech, language and head and neck cancer. All faculty members have primary or secondary appointments within the Department of Otolaryngology, creating an ideal environment for translational research between basic and clinician scientists. Through this multidisciplinary approach to research training for different levels of trainees from a variety of fields, recruitment and retention of a research-oriented academic faculty involved in research into disorders of the ears, nose and throat will be increased.

? DESCRIPTION (provided by applicant) Taste buds comprise 50-100 elongate taste cells which detect sapid molecules in the oral cavity. The apical pole contains the taste receptors, while the basolateral membranes contact sensory nerve fibers that transmit taste information to the brain. Taste buds contain three types of fusiform cells distinguishable by morphological, molecular and functional criteria. Of these, Type II cells are the best understood. They contain the taste receptors and downstream signaling effectors for bitter, sweet and umami taste stimuli and when activated, release ATP to activate purinergic receptors on sensory nerves. The Type I cells serve a glial like support function, expressing enzymes for uptake and degradation of transmitters, including a specific ectoATPase for degrading the ATP released from the Type II cells. The Type III cells are the most enigmatic. They are required for sour taste and some aspects of salty taste, but the receptor mechanisms remain elusive. Further, Type III cells are the only cells in the taste bud to form discrete synapses with afferent fibers, but neither the transmitter released, nor its cognate receptor on the sensory nerve fibers has been identified molecularly. We have found that a specific population of geniculate ganglion neurons, those that express the 5-HT3a receptor, selectively innervate the Type III taste cells of the anterior tongue. Using mice expressing GFP from the 5-HT3a promoter, we have shown that although 5-HT selectively stimulates the GFP-expressing ganglion neurons, 5-HT is not required for transmission of sour or salty taste to afferent fibers, suggesting other transmitter(s) and receptors are required. Although 5-HT is not crucial to neurotransmission in this system, the expression of 5-HT3a by those ganglion cells innervating Type III taste cells allows us to distinguish this population from other gustatory ganglion neurons. In Aim 1, we will utilize the Fluidigm C1, a new technology recently acquired by our Genomics and Microarray Core to generate and compare the individual transcriptomes of GFP-labeled (innervating Type III taste cells) and unlabeled geniculate ganglion neurons (which innervate Type II taste cells). This comparison will identify candidate neurotransmitter receptors expressed preferentially by the ganglion cells innervating sour- and salty-responsive taste cells. In Aim 2, we will use the bioinformatics information obtained from Aim 1 to test candidate transmitters on GFP- labeled ganglion cells in vitro to test functionality of the candidate receptor. Then we will utilize chord tympani nerve responses and corresponding antagonists to test functionality in vivo and the necessity for these receptors in transmission of taste information. Together the results will allow for the development of a new approach for studying synaptic transmission in taste buds as well as provide an important database for researchers studying proteins involved in signal recognition between taste cells and nerve fibers.

PROJECT SUMMARY Type III ?presynaptic? taste cells respond to sour and some salty stimuli with action potentials and release neurotransmitter at conventional synapses with afferent nerve fibers. The identity of the transmitters involved, however, is still unclear. We have shown previously that all taste stimuli (including sour and salty) require ATP signaling to neural P2X receptors for transmission to afferent fibers. Nonetheless, ATP release from Type III cells has not been detected. Conversely, Type III cells do release serotonin (5-HT) which activates 5-HT3 receptors on afferent nerve fibers but neither knockout nor pharmacological inhibition of 5-HT3 completely eliminates responses to acids or salts, suggesting other neurotransmitters are involved. One problem in studying transmission from Type III cells is that the conventional stimuli used to activate these cells -- acids, salts, and KCl -- all have non-specific effects on other cell types in the taste bud. To circumvent this problem, we employ an optogenetic strategy permitting direct stimulation of Type III cells with light rather than chemicals. We developed a mouse that expresses Cre recombinase selectively in type III taste cells relying on Pkd2l1 as a Cre driver. When these Pkd2l1-Cre mice are crossed with ?floxed? channelrhodopsin (ChR2) mice ChR2 is faithfully expressed in taste cells immunoreactive for PKD2L1 with no off-target expression in other taste cell types. Flashing 470 nm light onto the tongue elicits responses in the chorda tympani and glossopharyngeal nerves that are robust and repeatable, resembling responses to sour and salty stimuli. In this proposal we will utilize these Pkd2l1-ChR2 mice plus other gene-targeted mice to investigate the neurotransmitters that are used by these cells to communicate with the nervous system, and the perceptual qualities evoked by selective stimulation of PKD2l1 Type III cells. Aim 1 will investigate the role of ATP and other neurotransmitters (i.e., 5-HT, GABA, and NE) in activating geniculate ganglion neurons that selectively innervate Type III cells. Calcium imaging of isolated geniculate ganglion cells will identify transmitters that activate these neurons, and chorda tympani and glossopharyngeal nerve responses to light in the Pkd2l1- ChR2 mice will allow us to test the role of their cognate receptors in vivo. Aim 2 will address the perceptual quality elicited by stimulation of Pkd2l1-ChR2 cells, using behavioral paradigms, and cFos activation will be used to address the central representation of PKD2L1-expressing Type III cells. In Aim 3 we will test the hypothesis that PKD2L1-Type III cells participate in an intragemmal (intra taste bud) circuit, resulting in modulation of other taste qualities, particularly those transduced by Type II cells. Results from these studies will provide important new information about transduction, afferent neurotransmission, and intragemmal signaling in Type III taste cells.

Project Summary The broad goal of the proposed experiments is to identify key molecules that allow mammals to detect basic tastes and generate electrical responses that are conducted to brain regions. Molecular mechanisms of taste reception have been a subject of intense investigation over the last 30 years, with great strides made in identifying receptors for bitter, sweet and umami. Much less is known about the nature and function of receptors for sour, the taste that allows us to detect acids in spoiled foods or citrus fruits. In this proposal, we will begin to unravel this problem as we test the contribution of the newly discovered otopetrin proton channels in the transduction of acidic and ionic tastes. These experiments build on the progress made in the last grant application, where we used a combination of cellular, molecular and functional approaches to identify the pH sensitive ion channels in Type III taste receptor cells (TRCs) that mediate sour taste. Notably, we described a novel proton-selective ionic current that is likely to be a key component of sour taste transduction. In the last funding period, we successfully identified the gene that encodes the proton channel, through functional screening of genes enriched in Type III TRCs. Among 41 genes tested, we identified one, encoding the transmembrane protein Otop1 that upon expression induced a proton current in both Xenopus oocytes and HEK-293 cells. Interestingly, Otop1 was first identified as a gene mutated in mice with vestibular defects (?tilted? or tlt) but its function in the vestibular system and elsewhere in the body was not understood. Building on these new results, we propose three specific aims to test the role of the Otop channels in taste signaling. The first aim will examine the functional distribution of Otop1 across the tongue and palate epithelium, allowing us to answer the question of whether Otop1 is the sole ion channel mediating proton influx in the gustatory system. In the second aim, we will measure cellular responses to acids in wildtype and Otop1 KO mice in order to determine the degree to which Otop1 contributes to sensory responses, ex vivo. In the third aim, we will measure responses from gustatory nerves and assess behavioral thresholds for acid detection in wildtype and Otop1 KO mice to determine the extent to which Otop1 mediates responses to sour taste stimuli in vivo. Together our experiments will allow us to determine if Otop1 functions as a sour taste receptor. Our efforts to identify mechanisms of taste transduction may allow the development of taste modifiers that can be used to enhance palatability of food, reducing the need to add sweeteners that contribute to the development of diabetes or salts that contribute to hypertension. Moreover, the proposed experiments will provide basic information regarding the functional properties of this new family of proton channels that will help us understand their contributions to diverse physiological processes, including brown fat metabolism and the development and maintenance of the vestibular system.

Abstract Taste buds are multicellular sensors reporting taste, which reflects quality and potential toxicity for potential food items. Two of the 3 cell types (Type II and Type III) comprising a taste bud are well described in terms of molecular features for transduction and transmission of different taste qualities whereas Type I cells are described as being supporting or glial-like. In this proposal we test the hypothesis that Type I taste cells serve two critical functions. First, our preliminary reconstructions of taste buds show that Type I cells completely surround and separate other cell types in a taste bud; thus Type I cells must be important for signaling and integration of taste information within the bud. Second, our ultrastructural analysis further shows that some Type I cells extend an apical process through the taste pore to sample the oral contents and so may transduce some components of salty taste. We propose to use correlated anatomical, functional and molecular means to classify and characterize Type I cells in taste buds from mice. The first set of experiments will extend our preliminary analysis of Type I cells in circumvallate taste buds to test whether Type I cells in fungiform taste buds similarly separate Type II and Type III cells from one another. Further we test whether the Type I cells participate in intrabud signaling by releasing GABA or other neurotransmitters in response to the ATP released by stimulated Type II (sweet-bitter-umami) cells. The GABA can then act on adjacent Type II cells to terminate signaling and reduce subsequent responsiveness to tastants. In Aim 2, we test whether any morphological subtype of Type I cells expresses functional amiloride-sensitive salt (ENaC) receptors and if so, determine the relationship of these cells to sensory nerve fibers. Conventional EM indicates that Type I cells do not form synapses leaving open the question of how any Type I cell response could be communicated to the nervous system. We suggest that Type I cells, like glial cells in the CNS, can release neurotransmitter by reversal of membrane transporters for GABA or glutamate or opening of large pore channels. In the end we will have resolved two important questions in transduction and transmission of taste information by defining the nature of intrabud signaling between receptor cell types, and secondly finally determining the identity of cells underlying amiloride-sensitive salt transduction.