Robert M. Bradley - US grants

When horseradish peroxidase is injected into the circumvallate papilla on the posterior rat tongue, a small, discrete group of motor neurons is labelled in the brainstem. These neurons are located just ventral to the taste projection of the glossopharyngeal nerve in the nucleus of the solitary tract. This nucleus is analogous to, and an extension of, the dorsal motor nucleus of the vagus nerve; therefore, it is termed the dorsal motor glossopharyngeal nucleus. The objective of the proposed pilot, feasibility studies is to test the hypothesis that this nucleus projects to and controls secretion of the von Ebner's glands. The von Ebner's glands are minor salivary glands located beneath the circumvallate papilla, which have received little systematic investigation. Horseradish peroxidase will be injected into the dorsal motor glossopharygeal nucleus and the circumvallate papilla and von Ebner's glands subsequently examined for reaction product. Also, the nucleus will be electrically stimulated to determine whether this elicits secretion by the glands. These studies will identify the motor neurons controlling secretion of one set of salivary glands and will provide the basis for a comprehensive study to clarify the reflex control of salivation via taste stimuli.

The proposed research is to understand the extent of integration in the brainstem of sensory information from the posterior oral cavity and larynx. Receptors in these areas respond to thermal, chemical, and mechanical stimuli and initiate a number of upper airway protective reflexes, such as gagging, coughing, choking and swallowing. Sensory information from thse receptors travels in the glossopharyngeal and superior laryngeal nerves and projects to three areas with a known role in oral-laryngeal reflex control: the solitary nucleus, the trigeminal nucleus and an area in the pons. We propose to record neurophysiological responses from single neurons in each of these three brainstem projection areas while stimulating the epiglottis and posterior tongue with chemical, tactile and thermal stimuli. New information will be collected on the extent to which input from the two receptor areas converge onto single neurons, and on the breadth of responses of brainstem neurons to one or more stimulus modalities (taste, touch, temperature). Comparisons will be made of the response characteristics of neurons in the three brainstem areas. Experiments will be conducted in lambs since there is already a body of knowledge on the physiology of upper airway reflexes in these animals. These results will contribute to understanding how sensory information is integrated at the brainstem level to play a role in initiation and control of upper airway reflexes. Abnormalities of the triggering mechanisms of upper airway reflexes and aberrant neuromuscular patterns in the reflexes are serious clinical problems. The proposed research will provide new information related to the neural basis of these abnormalities and suggest improved methods of treatment.

DESCRIPTION (provided by applicant): In recent years gustatory research has made revolutionary advances at the level of transduction in taste receptor cells. However, the basic organization of the circuits responsible for processing this taste information at the brainstem gustatory relay nucleus has proved particularly difficult to analyze. The overall goal of this application is to better understand how the brainstem taste relay nucleus - the rostral nucleus of the solitary tract (rNST) - encodes, decodes and distributes the information it receives from the oral cavity. Specifically, differences in the morphology, neurophysiology, and synaptology of rNST neurons with known input and projection patterns will be determined. Such information is essential to understand sensory coding mechanisms in rNST because without knowledge of the underlying circuits it is problematic to make conclusions on how sensory information is processed. Most previous investigations of the rNST have relied on recordings from unidentified neurons with unknown roles in gustatory processing. However, there are reports that separate populations of rNST neurons project to either parabrachial or brainstem sites but not both. These separate groups of rNST neurons must have very different functional roles but since prior classifications of rNST neurons were based on whether they respond to lingual stimulation, they have all been uniformly classified as taste neurons and assumed to have similar functional roles in taste coding. Thus, the field is using a broad label of taste neuron for study of coding in rNST without in fact understanding the basic nature of the neurons and their connections. By specifically labeling rNST neurons with known projection patterns and known afferent input connections new information on rNST circuits will be determined. This approach is based on the hypothesis that different populations of rNST neurons that distribute chemosensory information to different brain areas have different neurobiological properties. The experiments described in this proposal will determine the degree to which the afferent information is transformed as it travels through the rNST and whether these different neuronal subsets have different synaptic characteristics. The results will provide necessary new knowledge for understanding how rNST neurons function in processing information derived from stimulating taste buds. PUBLIC HEALTH RELEVANCE In humans the sense of taste is a significantly factor in the quality of life. Taste stimuli guide food intake and contribute to the pleasure and drive of feeding behavior. Foods that have a pleasurable taste promote consumption and aversive taste leads to rejection. Thus, taste stimuli guide food selection. Recent scientific progress has characterized the receptor proteins and transduction mechanisms at the level of the taste receptor cells but the central nervous system circuits responsible for processing this taste information have received less attention. The overall goal of this application is to better understand the role of the rNST in processing gustatory information. Understanding the neurobiology of taste guided behavior relates to how humans make decisions about food intake which ultimately plays a critical role in human health.

microscopy; biomedical equipment resource;

The primary objective of this research is to develop an implantable recording electrode that will permit electrophysiological study of uncut, single taste fibers for long time periods in unanesthetized animals. An implantable electrode, that consists of a silicon chip with a sieve-like array of small holes and associated electrodes, will be placed in small tubes. The electrode and tube will then be surgically implanted in the neck of rats, with the cut glossopharyngeal nerve positioned in each end of the tube. Regenerated fibers grow through the holes in the silicon chip in apposition to electrodes. Subsequently, recordings will be made from single taste fibers in chronic preparations. Thus, techniques will be available to approach a fundamental question about the cell biology of the taste system: to what extent is the response from a single afferent fiber stable during the ongoing processes of taste bud cell turnover and synaptic remodeling? In addition we propose to use this implantable device to examine and analyze the response patterns across fibers to chemical stimulation of the tongue. These studies will contribute basic information about the stability of the single afferent response during processes of taste cell turnover and synaptic changes. This information will provide important new data about the cell biology of the taste system that can be applied to questions of neural coding in taste. In addition, development of the implantable electrode can have important applications in technology to restore function in regenerating peripheral nerves.

DESCRIPTION (provided by applicant): The rostral portion of the nucleus of the solitary tract (rNST) in the brainstem is the first central taste relay. The rNST receives primary afferent projections from facial and glossopharyngeal nerves, which innervate lingual taste buds and papillae and terminate centrally in a topography that reflects peripheral organization. The time course for and molecular factors involved in proliferation and differentiation of rNST cells are not well understood. Given the importance of an organized taste system for proper function, the long-term objective is to determine how the rNST is established in embryonic rat. The proposed research aims are to determine: the development of solitary tract (ST) projections; the developmental time course of proliferation and differentiation of cell components comprising the rNST; the development of biophysical properties and synapses of rNST neurons; specific molecular factors present in relation to rNST development; and, to manipulate the expression of these factors to determine effects on rNST development. Immunofluorescence will be used to identify the time course for development of neurons and glia that compose the rNST in staged embryos. An in vitro brainstem slice preparation will be used to determine development of neuron function. Immunohistochemical and Western blot approaches will be used to identify the expression of Sonic hedgehog (Shh) signaling factors potentially important for proliferation and differentiation in the NST, and cell cycle regulators across developmental stages. Finally, an explant culture system will be used to isolate the brainstem and manipulate in vitro Shh signaling molecules localized to the rNST and surround. The working hypotheses are that during embryonic development, neural precursors migrate from the fourth ventricle and differentiate to form the earliest rNST, where neurons differentiate before glia; that rNST neurons are functional, but with changing properties, in the embryo; and, that Shh signaling regulates the timing of proliferation and differentiation of rNST neural precursors and emerging neurophysiological function in rNST. Results of these experiments will provide an understanding of the early establishment, organization and function of the rNST and will demonstrate possible molecular pathways involved in that development. This will provide an important foundation for understanding the formation of the primary afferent relay of the gustatory system and lay the groundwork for understanding the demonstrated plasticity that occurs in this relay when environmental manipulations take place during embryogenesis. Furthermore, the proposal addresses essential questions in the process of neural patterning in the CNS.

DESCRIPTION (provided by applicant): Taste is a vital sense that depends on taste bud receptor complexes in the gustatory epithelia to direct eating and food choices. Taste bud cells and supporting epithelia turn over, are renewed throughout life, and are susceptible to environmental and pharmacological agents. Taste organs therefore depend on tightly regulated proliferation and differentiation. The Hedgehog (HH) pathway regulates maintenance of adult stem and progenitor cells in many tissues. Our data implicate HH signaling as a principal regulator of maintenance and renewal of taste receptor organs. However, HH activity not only regulates tissue maintenance, but also uncontrolled HH signaling is the cause of basal cell carcinoma (BCC), a common skin tumor. Therefore, HH Pathway Inhibitors (HPIs) that block signaling by affecting the HH pathway effector, Smoothened, have been developed as targeted therapeutics for BCC. HPIs lead to regression of BCCs, but patients often discontinue treatment due to adverse effects including severe taste disturbances. Our preliminary data suggest that the taste alterations are an on-target effect reflecting a strict requirement for HH signaling in taste function. We hypothesize that HH signaling functions to control renewal of taste organs and that pharmacological disruption of this control is responsible for chemosensory disturbances in patients treated with HPIs. We use genetic models (mouse) and pharmacological treatment (mouse and human cancer patients) to study the taste system with altered HH signaling. Our Multi PI approach includes chemosensory and HH signaling biologists, and a clinician/scientist treating BCC patients with HPIs. In Aim 1 we hypothesize that HH signaling regulates taste bud and/or papilla maintenance and function through an essential role in epithelial tissue renewal. In mouse we analyze: Hh pathway gene expression pattern and signaling in taste organs throughout the oral cavity; taste bud receptor cell maintenance, renewal and function, during and after treatment with HPIs that target the signal transduction component Smoothened; and, in genetic models, effects of targeted deletion of Smoothened on taste organs. We study cell and tissue effects, and behavioral and neurophysiological taste function. In Aim 2 we propose that HH signaling acts to control taste organ maintenance and function in BCC patients, explaining why pharmacological inhibition of this pathway causes chemosensory disturbance. In patients receiving HPIs, we test predictions about the extent and time course of chemosensory disruption, before, during and after HPI treatment, with questionnaires and NIH Toolbox tests of taste and smell sensory function; and, we quantify the number and distribution of fungiform papillae to correlate with taste sensation tests. The project addresses mechanisms of HH signaling inhibition in altering taste organ dynamics and function. This knowledge contributes to explaining the poorly understood, taste disturbances in patients treated with HPIs, and could ultimately lead to dietary modifications or other approaches to ameliorate chemosensory disruption and improve quality of life.