Robert E. Stewart, Ph.D. - US grants

DESCRIPTION: The goal of this research is to investigate the distribution of the epithelial sodium channel subunits involved in salt taste in relation to a and species differences in gustatory sodium sensitivity. Previous recordings from the whole chorda tympani (CT) nerve in rats and hamsters indicate opposit developmental patterns in gustatory sodium sensitivity -- young rats are more sensitive than mature rats, whereas young hamsters are less sensitive than mature hamsters. The first specific aim is to analyze the difference between young and adult hamsters using single fiber recording techniques. Complementa studies on the rat are currently in progress. The stimulation device allows chemical stimuli to be presented to taste receptors in a patch of lingual epithelium under voltage clamp while recording from a single chorda tympani nerve axon. Data on the voltage sensitivity of the CT response are then used derive estimates on the affinity and density of the epithelial sodium channel, as demonstrated in previously published studies. The hypothesis is that, compared to young hamsters, mature animals will show reduced sodium response frequencies, fewer fibers that are most responsive to sodium, and lower estimates of channel affinity and/or density. The second aim is to employ recently developed antisera to immunohistochemically identify the subunits of the epithelial sodium channel involved in taste transduction in young and matu hamsters and rats. Fluorescence microscopy will be used to examine the distribution of immunoreactivity within the taste buds to antisera for the alpha, beta, and gamma subunits of the sodium channel. The hypothesis is that the age and species differences in chorda tympani sodium sensitivity will be reflected in corresponding differences in the expression and distribution of t channel subunits.

DESCRIPTION: (Adapted from the Investigator's Abstract) The hamster is a popular model for taste studies and considerable effort has been focused on understanding the basic biological events that underlie taste stimulus reception in this species. However, our knowledge of the sodium and potassium sensing pathways in the intact hamster taste system is limited. The aims of this research are to extend our current electrophysiological and biophysical knowledge regarding sodium taste in mammals to the hamster, and to also provide information about the physicochemical basis of potassium ion sensing in this species. These finding will provide important insight about taste system function to researchers interested in stimulus coding strategies in the hamster taste system. Furthermore, the findings will be of fundamental importance to continued research directed at understanding mechanisms of functional regulation of sodium taste transduction elements. The pivotal role of the taste system in food selection makes its function an important clinical factor in diet-related health problems, including heart disease, diabetes, cancer and malnutrition. Therefore, in addition to basic information about gustatory function, the proposed research will provide information that may ultimately be of significant clinical value with respect to changing gustatory function in human development, health and disease.

DESCRIPTION (provided by applicant): The taste systems of several rodent species exhibit impressive morphological and physiological alteration during a prolonged postnatal period. In hamster, remarkable postnatal changes in sodium and saccharide sensing have been described, and preliminary evidence suggests that hamster bitter sensitivity also undergoes considerable developmental alteration. Evidence suggests that changes in bitter and sweet sensing could depend upon age-regulated expression of elements of the receptor/transduction apparatuses for these stimuli. Thus, the specific aims of the proposed research are to identify the temporal and spatial expression pattern of G protein-coupled receptors for sweet (and, time permitting, bitter) stimuli in developing hamster taste system primarily using in situ hybridization and confocal microscopy. The proposed research represents an initial exploration of the molecular basis for the postnatal ontogeny of physiological sensitivity to sweet taste stimuli. More fundamentally, it would provide additional insight regarding the molecular basis for taste sensing in this important rodent model of peripheral and central gustatory structure and function. The findings from the proposed work will be valuable in constructing a mechanistic understanding of age-related changes in taste stimulus sensing mechanisms. Such developmental changes in taste function may act to shape adult dietary predilections, and thus could influence diet-related risk factors in cardiovascular, renal, metabolic, and/or neoplastic disease. Therefore, the proposed work may also provide information of significant clinical value.

With this award from the Major Research Instrumentation (MRI) program, Drs. Watson, LaRiviere, Stewart, Erickson, Turner and ten colleagues from the Departments of Biology, Psychology, Physics and Engineering, and Computer Science, along with the Programs in Neuroscience, Biochemistry, and Environmental Sciences at Washington and Lee University (W&L), Virginia Military Institute (VMI) and Mary Baldwin College (MBC) will acquire an Olympus Fluoview 1000 (FV1000) Spectral Confocal Live Cell System for research and training across the sciences. This confocal laser scanning microscope (CLSM) will form the foundation of the newly created communal microscopy, imaging, and computational core of a projected Interdisciplinary Quantitative Science Center that is part of a long-range, grant-funded initiative at W&L which promotes mathematical modeling, bioinformatics, and data analysis. The confocal microscope will enable fifteen researchers from three schools to expand their ability to detect, quantify, and localize gene products and to study biological structures, thereby expanding the scope of existing research agendas and developing new as well as collaborative research opportunities. Specifically, the confocal microscope will facilitate and impact the following areas: 1) the study of different aspects of synaptic connectivity and influences on neuronal wiring in the brain and central nervous system, 2) eukaryotic nonfunctional ribosomal RNA decay pathways, 3) effects of hormone regulation in the developing cardiovascular system and sexual differentiation, 4) mechanism of Ca++ regulation, 5) reconstruction of glandular morphology of terrestrial invertebrates, 6) ultrastructural analysis of single-celled eukaryotes, 7) rapid assessment of otolith structure in new fish species, 8) comparison of the formation of dense core secretory granules cells, 9) localization of nitrogen-cycling microorganisms on freshly harvested fine roots and organic matter and 10) image segmentation and analysis studies.

Confocal microscopy is a technique that allows a three dimensional high resolution image acquisition of live or fixed specimens. By attaching fluorescent dyes (fluorophores) to biological specimens, cells and sub-cellular components can be identified with a high degree of specificity amid non-fluorescing material. Moreover, several target molecules can be visualized simultaneously with multiple fluorophores emitting light at differing wavelengths. Using image analysis software, the acquired serial optical sections are then rendered to generate a clean high resolution three-dimensional reconstruction of the specimen in which all out-of-focus light has been rejected. The result is an exceptionally clean high resolution image of a biological specimen that can reveal the presence of a single molecule. This instrument is critical for the study of biological structures and will have a transformative effect on how emergent young scientists are trained at W&L, VMI, and MBC. The CLSM will provide cutting edge tools for faculty to carry out their research projects and provide microscopy training and research opportunities to undergraduate students from three rural primarily undergraduate institutions. Using the teacher-scholar model, which seamlessly integrates research with discovery-based laboratory course work, W&L and VMI, will take full advantage of this instrumentation to promote further research integration into courses, to expand undergraduate programmatic research opportunities, and to promote student and faculty collaborations within and between departments and institutions.