Sidney Simon - US grants

The molecular events underlying chemical transduction in lingual epithelia are not well understood. In this proposal we will measure the electrical, chemical and structural changes that occur in mammalian lingual epithelia in response to chemical stimuli so that models of taste transduction can be tested or formulated as the evidence dictates. The recent discovery that the tongue actively transports ions (1) suggests that previous models of taste transduction that have assumed it to be impermeable to ions and that there are separate pathways for Na+, K+, and H+, and non-electrolytes may have to be modified. Here the active and passive transport properties of dog tongue will be studied by mounting it in a Ussing chamber. The role of taste cells and their proposed receptors in the transduction process will be studied by comparing the passive and active transport behavior of the tongue in the presence and absence of taste papillae. Morphological and intracellular ion changes will be measured using both conventional electron microscopy and electron probe microanalysis. These data will then be used to formulate mechanisms of taste transduction for salts, acids, D and L amino acids, and saccharides. In major goal of this research is to describe the dog tongue as a transporting epithelium. The transport pathways for mono and divalent anions and cations, saccharides, acids, and amino acids will be determined as well as their selectivity isotherms. The effect of ADH, copper, D-penicillamine, nystatin, amiloride, ouabain, C-AMP, intracellular Ca++, and site-specific reagents known to eliminate specific taste responses will be tested for the purpose of determining rate-limiting steps of transport, eliminating particular transport pathways, to determine those cell types that respond to various chemical stimuli, and to determine the role of various hormones in the transduction process. The data we obtained on the voltaged clamped preparation will be correlated with taste perception data which to date have been in substantial agreement. This voltage-clamped epithelial preparation can be used to assay the effect of drugs used to treat patients with taste disorders (such as hypogeusia, which arise at low copper concentrations) and also as a bioassay to identify new artificial tastants.

Cellular changes in fungiform papillae from mammalian tongue resulting from degeneration and subsequent regeneration of the chorda tympani nerve bundle (CT) will be measured. We propose and will test a hypothesis that predicts that upon transection of the CT the initial degeneration (and subsequent regeneration) of the taste buds is correlated with the ability of the epithelial cells to maintain (or lose) their intercellular contacts (i.e., tight junctions, desmosomes, gap junctions). Epithelial transport measurements for different tastants and elicited responses from the CT will be used to demonstrate that unilateral transection of the CT has pronounced effects on both the ipsilateral and contralateral sides of the tongue. After unilateral CT transection epithelial responses to salts (NaCl, KCl), saccharides (sucrose, D- glucose), acid (HCL) and a bitter tastants will be measured as a function of time to determine if the transport pathways for different tastants are expressed at the same or different times. Cellular processes involved in taste transduction will be elicited by measuring the transport pathways for ions and nonelectrolytes through isolated dog and rat tongue. Patrticular emphasis will be placed on characterizing the entry and exit pathways for ions that are activated by the presence of various salts and sweet and bitter tastants under a range of ionic conditions. The saccharide stimulated, amiloride inhibitable transport pathway will be studied in detail by obtaining its ion selectivity as well as salt and voltage dependence. The effects of c-AMP on the stimulation of transport by bitter tastants, caffeine and quinine will be studied on regions of tongue having fungiform and/or circumvallate papillae.

The overall goal of this research is to determine the cellular mechanisms responsible for chemical oral irritation. This will be achieved by obtaining single unit recordings from rat lingual and chorda tympani nerves, and patch-clamp recordings from rat trigeminal ganglion neurons in response to chemical irritants. The irritants include those found in spices such as red and black pepper, horseradish, ginger, mustard, cinnamon, and onions; and in pollutants such as acids, alcohols, aldehydes, and acetates. When placed on lingual epithelium, these compounds produce a variety of sensations that range from warm to burning to painful. Emphasis will be placed on determining the neural responses elicited by capsaicin, the pungent ingredient in ed pepper that has been shown to activate a subset of pain fibers. Capsazepine, a specific capsaicin receptor antagonist, will be used to determine whether responses occur through capsaicin- sensitive pathways. Olvanil and tinyatoxin will be used as non-pungent capsaicin analogues. The specific aims include determining: 1) whether irritation is caused only by the activation of a subset of lingual nerve pain fibers, 2) whether irritants activate chorda tympani fibers, 3) whether activation of lingual nerve fibers is a consequence only of the irritants' activation of capsaicin receptors, 4) whether the irritant-induced currents in isolated trigeminal ganglion cells are inhibited by capsazepine, 5) whether a specific current is associated with pungency, and 6) whether irritants can modulate chorda tympani responses to taste stimuli. This research will provide fundamental information relating to taste, irritation, and pain. Regarding issues of health, capsaicin and its analogues are used clinically in relieving trigeminal neuralgia and vasomotor rhinitis as well as other painful symptoms. Understanding the basic mechanisms of how irritants activate nerves could lead to the development of effective countermeasures against irritating stimuli.

Eight investigators from five departments of Duke University will purchase an isothermal titration calorimeter. This instrument will provide the investigators with access to a state-of-the-art isothermal calorimetry instrument which will allow elucidation of the fundamental thermodynamics of molecular recognition processes in research currently underway in the laboratories of the investigators. Among the systems to be studied are: (1) protein - ligands and their interactions; (2) signal peptides in lipid bilayers; (3) carbonic anhydrase and metal affinity; (4) extracellular matrix proteins and cell receptors; (5) membranes and macromolecular binding; (6) interactions of ATP and other agents with DNA topoisomerase; and (7) neurotrophins in cortical neurons. The new instrument, in conjunction with ongoing structural and biochemical studies will permit a molecular level dissection of structure-function relationships in these important systems.

DESCRIPTION: (Adapted from the Investigator's Abstract) Neural activity from receptors in the anterior tongue gives rise to a gustatory message that reflects the activation of both the taste and trigeminal somatosensory pathways. Currently not understood, and is the focus of this application, are the peripheral and central mechanisms regarding how the trigeminal (TG) system influences the taste system. The overall goals in this proposal are (1) to determine how compounds that activate TG neurons can modulate responses evoked from taste receptors cells (TRCs) and chorda tympani (CT) neurons, and (2) to quantify the contributions of the trigeminal somatosensory (mechanosenosry and nociceptive) pathways to the encoding of gustatory information in the insular cortex. It is proposed that, when activated, TG neurons will release neuropeptides, such as substance P and CCK, that will bind to receptors on TRCs and thereby modulate them. That in turn will modulate the activity of CT neurons to lingual applications of NaCl, acid, sucrose, and quinine. This hypothesis will be tested by activating the TG nerve electrically and chemically (e.g., with capsaicin, the pungent ingredient in hot pepper) and also by comparing CT responses before during and after the system injection of agonists and antagonists of substance P and CCK. A variety of methods (RT-PCR, immunocytochemistry, patch clamp, and measuring changes in intracellular calcium) will be used to determine whether CT modulation by TG activators such as capsaicin, is direct (on TRCs) and/or indirect via peptide release. Emphasis will be on identifying receptors for neuropeptides, capsaicin (VR1), and proton-gated channels in TRCs, lingual epithelium and geniculate ganglion neurons. To quantify the contribution of the TG somatosenory pathways to the cortical encoding of gustatory information, bundles of microelectrodes will be implanted in the primary gustatory (insular) cortex. Then, in lightly anesthetized rats, the simultaneous responses of populations of single insular neurons to a set of standard tastants will be measured before and after the selective inactivation of TG ganglion innervating the anterior tongue, a procedure that will simulate lingual nerve injuries. In addition, cortical-cortical influences on gustatory coding will be tested by reversibly inactivating the primary somatosensory cortex (SI). Information obtained from both the peripheral and cortical recordings will provide novel molecular, cellular and circuit mechanisms regarding the modulation of sensory information, and quantitative information regarding the contributions of the TG system to gustatory coding.

Increases in pulmonary related incidents of morbidity and mortality have been epidemiologically associated with exposure to particulate matter (PM). Due to its heterogeneity and complex physicochemistry, the underlying mechanism(s) of PM toxicity are poorly understood. This proposal examines the hypothesis that physicochemical components of PM initiate inflammation by activation of irritant (i.e., capsaicin, acid-sensitive) receptors located on sensory nerve endings and airway epithelial target cells. This activation results in an influx of extracellular calcium and release of neuropeptides and inflammatory cytokines, which proceed to initiate and sustain events of airway inflammation ands hypersensitivity. The response of irritant receptors to PM will be examined in primary cultures of sensory dorsal root ganglion neurons and in human bronchial epiothelial cells. Biophysical (e.g., patch clamp, calcium imaging) and immunological (e.g., cytokine release) endpoints will be used to characterize the response to various urban, industrial and ambient PM. The physicochemical properties of selected PM and their separate soluble and particulate fractions will be determined and examined for their cellular effects. Based on these data, synthetic particle analogues will be designed, that resemble PM particles in size and surface charge, to assess the inflammatory contribution of specific PM components. Physiological and pharmacological evaluation of the responses to PM will identify and characterize the contribution of culpable irritant receptors to the PM-mediated effects. In addition, the involvement of other cellular pathways (i.e., calcium channels, acid-sensitive exchangers) will be examined. The mechanistic approach outlined in this proposal will identify the responsible factors and target sites contributing to PM toxicity.

DESCRIPTION (provided by applicant): The sense of taste enables an organism to identify potential sources of food. Whether the organism ingests the food depends on its palatability as well as the homeostatic state of the animal. Gustatory processing involves the primary gustatory cortex (GC), the orbitofrontal cortex (OFC), the amygdala (AM) and the hypothalamus (HT). These interconnected brain areas are involved in processing a tastants' identity and its reward value as it pertains to the animal's motivation to eat. We have developed the technology to simultaneously record the activity of neuronal ensemble in all four of these areas from rats with chronically implanted electrodes that tick to obtain rewarding tastants and whose motivation, as it pertains to their state of hunger or satiety, can be modulated by eating or by peptides that will enhance or suppress their appetite. At present there is a paucity of information regarding how the interactions in and between brain areas of the taste-reward circuitry change as an animal eats to satiety or is made hungry or sated by the injection of appetite-modulating peptides. Our goal is to elucidate how the processing of tastants is distributed among these areas when an animal freely licks to receive tastants and when its motivation to eat changes. Our first aim is to test the hypothesis that the neural activity obtained from populations of GC and OFC neurons can be used to: discriminate among tastants in a single lick, anticipate the tastant that will be delivered when it is expected, and predict when an animal will begin and terminate drinking. The second aim is to show how changes in motivation, as it relates to hunger and satiety, affect the processing of gustatory information throughout the taste-reward circuitry. This information will be obtained by simultaneously measuring the changes in neural activity that occur in and between the GC, OFC, AM and HT as an animal voluntarily begins feeding various tastants to satiety, and by manipulating its state of hunger or satiety through the central administration of appetite- suppressing (leptin, cholecystokinin), or appetite enhancing (neuropeptide Y, orexin, ghrelin) peptides. We will test the hypothesis that eating to satiety as well as the injection of these peptides will cause distinct changes in neural activity that will be observed throughout the circuitry and that information on the physiological state of the animal (i.e. hunger, satiation) can be more accurately obtained if neuronal information from the above mentioned brain areas are acquired simultaneously. Obtaining such knowledge is relevant to the important societal problem of obesity, which has reached epidemic proportions. In summary, we will use a novel preparation that takes advantage of state-of-the-art technology to address fundamental scientific and public health issues regarding how behaving animals process gustatory information across different motivational states.