Nirupa Chaudhari - US grants

Calcium channels regulate a wide range of cellular activities. Based on physiological and pharmacological evidence, diverse calcium channel types exist and are presumably derived from independent genes. The regulation of these calcium channel genes during normal development and their effects on other genes are poorly understood. This project utilizes naturally occurring mutations in the calcium channel genes of vertebrates to gain insights into the structure of calcium channels and the modifications in electrophysiological and cellular functions that ensue from the altered structures. Muscular dysgenesis (mdg) of mice is a mutation in the gene for the alpha 1 subunit of the skeletal muscle dihydropyridine (DHP) receptor and leads to loss of normal calcium channels and excitation-contraction coupling. However, dysgenic muscle does contain a low level of a mRNA hybridizable with DHP receptor alpha 1 cDNA and also expresses an unusual calcium current. Detailed molecular characterizations (via cDNA cloning) of this DHP receptor mRNA from dysgenic muscle will be carried out to obtain insights into the structural domains within the normal DHP receptor which give rise to its electrophysiological properties. Expression of the "dysgenic mRNA" in heterologous systems and antisense RNA inhibition of expression in dysgenic myotubes will be used to establish definitively whether this mRNA encodes a novel calcium channel. The crooked neck dwarf (cn) chicken and the cardiomyopathic (cm) hamster are known to exhibit functional alterations in their calcium currents. These mutants strains will be investigated along similar molecular genetic lines to determine if the functional alterations arise from mutations in the calcium channel genes in these mutants also. Finally, the effects of the DHP receptor on the accumulation of other mRNAs involved in muscle differentiation will be examined in normal myotubes developing under experimental paralysis in culture. These multi-disciplinary and collaborative studies will provide valuable new insights into the structure-function relationships, and the regulation, of calcium channel genes.

Unlike vision and olfaction, taste has not been examined extensively with the modern tools of molecular biology. This Program Project brings a synthesis of molecular biology, cell biology and physiology into the field of taste by analyzing glutamate receptors in taste buds. Glutamate is an important taste stimulus (e.g. monosodium glutamate, MSG). Responses to glutamate have been recorded in sensory fibers and from the brain. Psychophysical and hedonic aspects of glutamate have been researched at great length. Yet, the initial events in glutamate taste, namely the interaction of glutamate with membrane-bound receptors, remains relatively unstudied. Consequently, critical information about glutamate receptors in taste buds--their molecular structure, their localization in taste cells, their function and modulation--is missing. The experiments outlined in this Program Project will provide these important data and will identify the first specific receptor for a taste stimulus. In the long term, this will pave the way for a comprehensive definition of the entire peripheral sequence of events in taste reception--from ligand binding to signal generation in the sensory nerves. The findings will also increase our understanding and awareness of the controversial food additive, MSG. The unifying premise underlying our Program Project is that receptors that transduce the taste of glutamate in taste cells are similar to glutamate receptors in the brain. Brain glutamate receptors form 2 large extended families of ionotropic and metabotropic receptors. We propose to take advantage of recent information about the molecular biology of these receptors to investigate glutamate receptors in taste buds. We will conduct PCR (polymerase chain reaction) with degenerate primers based on brain receptors to search for novel glutamate receptors in taste buds. Selected PCR products from lingual tissue will serve as probes to isolate full-length cDNAs encoding taste-specific glutamate receptors (Chaudhari). We will use in situ hybridization to localize mRNAs to specific taste cells and immunocytochemistry to localize receptor proteins (Roper). We will conduct functional studies of glutamate receptors endogenous to taste cells using microelectrode recording techniques. We will also express cloned glutamate receptors in oocytes and in heterologous mammalian cells and use patch clamp techniques to measure currents elicited by glutamate under different experimental conditions (Kinnamon). Lastly, we will use conditioned taste aversion to investigate the significance for taste of pharmacologically distinct receptors (Roper). By integrating these three approaches in a small, focused Program Project, we will take full advantage of the expertise and enthusiasm of 3 independent, productive, and highly interactive principal investigators. The collective insights gained on the molecular physiology of glutamate in taste will be much greater than could be achieved from the individual efforts of these researchers.

DESCRIPTION (provided by applicant): The peripheral end organs for gustation are taste buds, which transduce information on the quality and concentration of chemical taste stimuli into a coded pattern of activity in postsynaptic afferent nerve fibers. In most neurons, transmitter release at presynaptic terminals is dependent upon voltage-gated calcium channels (VGCCs). Some taste stimuli are known to cause depolarization of taste cell membranes followed by Ca++ entry. Other stimuli apparently do not lead to membrane voltage changes. The goal of this new research program is to begin to address these critical last steps of information processing in taste receptor cells. Specifically, we propose to analyze voltage-gated calcium channels in taste cells. These channels are critical for the function of most neuronal synapses but have not been examined systematically in mammalian taste cells. The Specific Aims for the proposed research are: 1) To determine the molecular identities of voltage gated calcium channels present in taste buds. This aim will be carried out using reverse transcriptase-polymerase chain reaction (RT-PCR) on a mixed population of mouse taste buds isolated from circumvallate, foliate and fungiform papillae and the palate. Primer pairs used will be specific for each of the 10 known calcium channel alpha1 subunits (which form the channel pore and determine major functional properties) and for accessory beta, gamma and alpha2-delta subunits, all of which alter important functional properties, including sites for modulation by second messengers. 2) To determine whether the calcium channel types identified in Aim 1 correlate with taste specificities. We will search for co-localization of alpha1 subunits with key proteins involved in taste transduction. 3) To image voltage-gated calcium channel activity in taste receptor cells and determine if channel function is subject to modulation by second messengers relevant in taste transduction. Through these aims, we hope to gain a novel perspective on voltage-gated calcium channels, which play critical roles in all neuronal systems, but have been minimally studied in taste cells to date.

[unreadable] DESCRIPTION (provided by applicant): Recent studies have identified G protein coupled receptors (GPCRs) that respond to umami, bitter and sweet taste stimuli. Downstream signaling pathways for these GPCRs are beginning to be understood through powerful combinations of biochemical and genetic analyses. With the advances, have come significant discrepancies between physiological/behavioral analyses and molecular studies, especially for umami taste. The mechanisms underlying sour taste are far less understood, and many candidate transducer channels remain as candidates. How taste cells process taste signals and transmit information to sensory afferent fibers is virtually unknown. A critical discrepancy exists between physiological evidence that taste cells respond to multiple taste qualities, and molecular evidence that taste cells appear to express GPCRs for only one quality. The present application addresses these key open questions using newly developed methods to examine the gene expression profile of functionally defined taste cells. We hypothesize that signals from receptor cells converge onto a separate class of output cells within taste buds; only output cells form synapses with sensory afferent fibers. Critical tests of this hypothesis may resolve the current controversy on the breadth of tuning of taste cells. For umami and acid tastes, we will carry out functional imaging on isolated taste cells, using criteria derived from detailed studies in the slice preparation. Such functionally defined taste cells will then be subjected to single-cell RT-PCR and/or differential library screening to identify molecules associated with the functional phenotype. To test our hypothesis on output cells, We will employ mice in which functional cell lineages for cells (a) that express PLCb2 or (b) that synthesize biogenic amines are transgenically labeled with Green Fluorescent Protein (GFP) or b-galactosidase. Functional in situ imaging of taste cells from these mice will allow us to test whether there is a separate category of taste bud output cells, akin to ganglion cells in the retina. Differential library screening will then allow us to begin defining the functional relationship between receptor and output cells. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Owing to a worldwide epidemic of obesity, there is enormous interest in understanding physiological mechanisms that regulate body weight. Sweet taste sensitivity is likely to play a significant role in food selection, calorie balance and the onset and progression of disorders such as type II diabetes and obesity. In recent years, taste research has focused on the identity of sweet taste receptors, T1R2+T1R3, and their binding sites for sugars and other natural and synthetic sweeteners. Yet, the downstream transduction events within taste cells following sweet receptor activation are incompletely understood. And, mechanisms modulating the primary sensory signal to produce adaptation are unexplored. In this competing renewal, we will extend studies begun during the previous funding period on mechanisms of sweet transduction. These provide a foundation for understanding the interplay of signaling pathways for sweet taste. In particular, we will focus on the role of cAMP in both transduction and adaptation for sweet stimuli. We will achieve this through the use of a novel transgenic mouse, that we developed, that expresses an inducible fluorescent reporter for cAMP in selected populations of cells. Functional studies on taste buds will include real-time imaging for cAMP in individual taste cells, patch-clamp recordings, and Ca2+ imaging (taste buds that are either isolated from the tongue, or retained in a semi-intact preparation). These will reveal cellular functions in individual taste cells as they respond to sucrose and synthetic sweeteners. We will answer the following questions in two specific aims: 1. Is cAMP modulated in sweet-sensitive taste cells? Our transgenic, inducible cAMP reporter will allow us gain spatial and temporal resolution of cAMP modulation in mammalian taste cells. 2. How is the cAMP signal produced and what is its downstream consequence for sweet sensing? We will test whether the cAMP opposes, complements, or refines the well-characterized Ca2+ signal, and whether cAMP plays a role in sweet taste adaptation. These studies will provide important new information about the relative roles of cAMP and phospho- inositide signaling in sweet taste transduction and adaptation, and should provide a foundation for future studies on the role of sweet taste in obesity.

DESCRIPTION (provided by applicant): Many neuroactive peptides, acting at hypothalamic nuclei, are critically important central regulators of food intake via the gut-(blood)-brain axis. Oxytocin is a potent anorexigenic peptide that plays a key role at hypothalamic and hindbrain centers that regulate appetite, satiety and ingestion. Taste strongly influences food selection. Yet, the role of taste has not been integrated into models of appetite and satiety. The premise of this application is that oxytocin, in addition to its central effects, also modulates the peripheral taste signal. Oxytocin knockout mice show altered taste sensitivity and preference, specifically for sweeteners. Our preliminary data show that a membrane receptor for Oxytocin (OxtR) is expressed in a discrete subset of cells within the taste bud, and responds to physiological concentrations of Oxytocin. Based on published reports and our preliminary data, we hypothesize that oxytocin acts on glial-like cells within taste buds, and secondarily alters the sweet-selective sensitivity and output of taste buds. We will test this hypothesis through the following Specific Aims: 1: Which taste cells express oxytocin receptor? Using single cell gene expression profiling and immunocytochemistry, we will test this because cell type has implications for function. We will conduct these analyses in wild-type and transgenic mice, PLC22-GFP, GAD-GFP and OxtR-YFP. 2: Is OxtR functional in taste buds and does it influence taste-evoked responses? We will extend our preliminary Ca2+ imaging experiments on taste cells from OxtR-YFP reporter mice. Using laser scanning confocal Ca2+ imaging on a lingual slice preparation, we will evoke responses to taste compounds, and ask whether oxytocin modulates sweet (or other) taste evoked responses. We will also examine if taste-evoked secretion of the afferent transmitter, ATP, is modulated by exposure to oxytocin. These experiments will be carried out on taste tissue from OxtR-YFP and PLC22-GFP. All proposed methodologies to achieve these aims, although highly technical, are routinely performed in our laboratories, and are in our publications. This assures the feasibility of the project. We intend to use this Exploratory Research grant (R21) to launch a new area of investigation that may have significant translational impact. Understanding how peptides (especially those implicated in central pathways for satiety) influence the peripheral taste signal may suggest new avenues to address eating disorders. PUBLIC HEALTH RELEVANCE: Several peptides in the brain and gut regulate food intake and are intensely researched. Their malfunction results in overeating or anorexic behaviors, and/or changes in body weight. Ironically, the most intuitive driver of feeding, taste, has not been investigated as a contributor to appetite regulation. We present evidence and propose to study further, how the taste system may be modulated by at least two of the same peptides that control satiety in the brain. This understanding would suggest new pharmacological possibilities to address eating disorders.

DESCRIPTION (provided by applicant): Recent findings indicate that taste buds are much more complex than previously held. Mature taste cells display diverse properties and may communicate among themselves. There are suggestions that chemo-sensory detection by taste receptors is followed by a shaping of the sensory signal within the taste bud prior to transmission to the brain, much as occurs in other sensory system. Yet, the significance of cellular heterogeneity and intercellular signals within the taste bud remain to be explained. We will ask the following questions focused on the most prevalent cells in taste buds, the glial-like Type I cells: 1. Do Type I taste cells regulate the ionic environment in taste buds? Spatially buffering K+ is a key function performed by astrocytes in the CNS. Our data suggest that ROMK and certain other apical channels may form such a K+ clearance pathway in taste buds. We will use confocal Ca imaging of taste buds in lingual slices, afferent nerve recordings and behavioral assays to test the effects of K+ accumulation using physiological, pharmacological, and genetic manipulations of ROMK and other regulators of K+ efflux. 2. Do Type I taste cells use GABA as a gliotransmitter to modulate taste signals? Our pilot data suggest that Type I cells are key players in GABAergic circuits in taste buds. We will complete production of a unique transgenic mouse strain in which yellow fluorescent protein (YFP) is expressed only in Type I cells of taste buds. We will then use these and our other transgenic mice to identify the cell-type selective pattern of receptors and confocal Ca2+ imaging to analyze GABA-mediated responses in these cells types, especially with respect to the impact on taste-evoked responses. 3. Do Type I cells arise from local epithelium and what is their lifespan? Cells in taste buds have an estimated average lifespan of 10-14 days. Earlier studies did not confidently resolve taste cell types. We will employ a new technology for birth-dating cells to assess if the different taste cell types have different lifespans and lineages. Through this series of testable hypotheses and powerful new technologies focused on Type I taste bud cells, we will begin to address the larger, and decades-old question: why do chemosensory taste cells form communities (i.e. taste buds)? What signals are processed within taste buds and what role in taste reception does ongoing communication among the cells serve?

DESCRIPTION (provided by applicant): This multi-PI R21 proposal describes a high-risk, high-impact project that introduces a novel method for recording taste-evoked activity in gustatory afferent neurons. The work represents a major new direction for the PIs as well as for the chemical senses community, and thus is ideally suited for the R21 mechanism. The project will introduce a novel recording technique to produce new data on the taste sensitivities and transmitter systems of large ensembles of geniculate ganglion neurons. We will employ a new strain of genetically-engineered mice that express a fluorescent functional reporter, GCaMP3, selectively in sensory (including gustatory) ganglion neurons. We propose to develop a new recording technique to image taste-evoked activity in fluorescent geniculate ganglion neurons in anesthetized mice. Geniculate neurons will be imaged with scanning laser confocal microscopy and changes in fluorescence quantified to simultaneously measure activity in large ensembles of neurons with single cell resolution. Ganglion cells will be excited by prototypic sweet, bitter salty, sour, umami and fat taste stimuli, delivered in the oral cavity. We will measure the breadth of tuning, concentration-response relations and entropy for gustatory geniculate ganglion cells (Aim 1). Functionally characterized neurons will be isolated and single cell RT-PCR will be carried out to examine the neurotransmitter systems employed by functionally distinct taste neurons (Aim2). Successful completion of the two aims will have 4 outcomes: first, we will have developed a completely new method for recording afferent sensory activity in large numbers of gustatory neurons~ second, we will accumulate a large database of response profiles for gustatory afferent axons, providing a comprehensive catalog of their breadth of tuning and entropies~ third, we will determine whether different classes of sensory neurons (generalists~ specialists~ sweet-, sour-sensing, etc.) have different molecular expression profiles, such as distinctive transmitter systems, transcription factors, and so forth~ and finally, whether there i a unique class of fat-sensing geniculate ganglion neurons. The data will have implications for how taste is coded by sensory afferents (e.g., labeled line vs, combinatorial coding) and will tremendously increase our understanding about how sensory neurons process gustatory signals.

DESCRIPTION (provided by applicant): We have developed an innovative method to record taste-evoked activity in gustatory afferent neurons with good cellular and temporal resolution. The method uses confocal functional calcium imaging and transgenic mice that express GCaMP3 in sensory neurons. By carefully exposing geniculate ganglia in living, anesthetized GCaMP3-mice and applying taste stimuli to the oral cavity, we can now record robust and reliable responses to discern the principles behind the transmission of gustatory evoked signals from taste buds to the hindbrain. These data will considerably extend single unit electrical recordings from the chorda tympani and greater superficial petrosal nerves or from geniculate ganglion cells. We are able to recording simultaneously from large ensembles of neurons. We have also designed a powerful strategy to relate the functional response profiles of individual geniculate ganglion neurons to their patterns of gene expression and establish robust molecular markers for separate functional classes of neurons. In a concerted effort from two well-established laboratories, we now propose a multi-PI project to exploit this novel preparation and to answer key questions regarding taste. Our new approach will tremendously expand our knowledge of peripheral sensory processing in taste. Our preliminary data demonstrate the feasibility of all 4 tightly-focused Specific Aims: Aim 1: How do gustatory sensory afferent cells respond to sweet, salty, bitter, sour, umami tastes and fats? Aim 2: Do mixtures of taste stimuli enhance responses from gustatory sensory afferent neurons? Aim 3: Do ganglion neurons that express certain transmitter receptors innervate specific taste cells? Aim 4: Are there dedicated neurons that detect each taste stimulus, and if so, can specific molecular markers be identified that associate with gustatory afferent neuron responses for each taste quality?

PROJECT SUMMARY The afferent sensory neurons that innervate taste buds have eluded definition at the molecular level until very recently. Specifically, it has been unclear if separate classes of neurons innervate the well-established receptor cell types in the taste bud. It has also been unclear whether there are dedicated neuronal circuits that connect peripheral receptor cells, afferent sensory neurons and the first relay neurons in the Nucleus of the Solitary Tract in the brainstem. Progress has been impeded by the lack of molecular markers to distinguish gustatory neuronal cell types. We have recently completed a study on single-cell RNAseq of geniculate ganglion neurons. Our detailed bioinformatics analyses, which are publicly accessible, revealed neuronal subtypes based on deep separations in their transcriptome profiles. Our study also yielded markers which permit us to distinguish the classes of gustatory neurons. We propose to examine the three main classes, T1, T2 and T3, in several ways. We will assess whether each class of neurons associates with taste buds in a given receptive field, or with particular taste bud cells types. We will also examine the central projections of neurons of each class for insights into their potential roles in gustation. Second, we will evaluate whether the gustatory neuron types preferentially convey information on particular taste qualities. And finally, we will examine the significance of the novel hybrid gustatory-mechanosensory neuron type that we recently reported, using in vivo confocal Ca2+ imaging in transgenic mice expressing the Ca2+ indicator, GCaMP. Together, these analyses will be the first to assess the extent of heterogeneity in the peripheral and central connectivity and functional properties of defined subtypes of gustatory neurons.

Project Summary Single-cell RNA sequencing (scRNAseq) has made unprecedented strides in discovering previously unrecognized diversity of neuronal cell types and their functions. Using scRNAseq, we showed that the mouse geniculate ganglion contains 3 molecularly distinct types of gustatory neurons that innervate taste buds, T1, T2, and T3, each with unique patterns of gene expression. In a concerted effort from two well-established laboratories, we now propose a multi-PI project to test hypotheses regarding distinct functions for each of the major types of neurons and their subtypes. Our ultimate goal is to produce an integrated molecular and functional categorization of gustatory neurons similar to what has been so powerfully effective in the auditory, visual, and somatosensory systems. We propose using a newly-optimized method for in vivo confocal Ca2+ imaging, neuron-selective fluorescent markers, and chemogenetic silencing to reveal the functions of T1 and T3 geniculate ganglion neurons. Specifically, using GCaMP-based Ca2+ imaging, we will test the hypothesis that within the cluster of T1 neurons there are subclasses that respond to distinct taste qualities whereas neurons within the T3 cluster respond to multiple, convergent tastes. We will further test the hypothesis that T1 and T3 neuronal subtypes contribute to separate central pathways serving a number of taste-dependent functions downstream of initial detection in the taste periphery. Completing the above aims will move the field of taste into a new era of molecular-functional integration. Our findings will assist electrophysiology and circuit tracing studies in taste, will shed light on the controversy over labeled lines versus combinatorial taste coding, and will bring new information on gustatory neural pathways that are so important to nutrition and ingestive behavior.