David V. Smith - US grants

DESCRIPTION: (Adapted From The Investigator s Abstract): Recent studies have provided insights into the transduction of sodium salts and acids by taste receptor cells and the organization of chemical sensitivities in neurons of the afferent gustatory pathway. Information from an amiloride-blockable sodium channel projects into NaCl-best afferent fibers in the chorda tympani nerve and into both NaCl- and sucrose-best neurons in the nucleus of the solitary tract (NST) of the hamster. Human psychophysical studies have shown that this amiloride-sensitive transduction component occurs also in humans but contributes less to the NaCl response than it does in rodents. Blocking this component with amiloride in humans does not reduce the saltiness of NaCl or other salts; rather, it blocks their sour side taste, which is of considerable magnitude for LiCl and Na-gluconate. Adaptation to NaCl, on the other hand, eliminates the saltiness of all salts in human studies and reduces the response of all NaCl-sensitive NST neurons. Studies in this proposal address the relationship between human taste perception, the physiology of the hamster gustatory pathway, and hamster taste receptor mechanisms. The findings will provide a basis for interpretation of adaptation and cross-adaptation effects on hamster NST cells and in human psychophysical studies and will support the overall hypothesis that the cross-adaptation procedure reveals receptor commonalities. tract (NST) of the hamster. Human psychophysical studies have shown that this amiloride-sensitive transduction component occurs also in humans but contributes less to the NaCl response than it does in rodents. Blocking this component with amiloride does not reduce the saltiness of NaCl or other Na+ or Li+ salts; rather it blocks their sour side taste, which is of considerable magnitude for LiCl and Na-gluconate. Adaptation to NaCl, on the other hand, eliminates the saltiness of all salts in human studies and reduces the response of all NaCl-sensitive NST neurons. Studies in this proposal address the relations among human taste perception, the physiology of the hamster gustatory pathway, and hamster taste receptor mechanisms. The first specific aim involves human psychophysical studies that are designed (1) to show that the reported effects of amiloride on saltiness are a result of psychophysical methods that force subjects to combine estimates of separate sensations; (2) to investigate the contribution of an amiloride-sensitive transduction component to the sourness of acids; and (3) to demonstrate reciprocal cross-adaptation effects on the salty taste of NaCl and other Na+ and non-Na+ salts. The second specific aim uses single-neuron recording from the hamster NST to investigate (1) the effects of amiloride on the neural processing of the taste of acids and (2) the reciprocal cross-adaptation effects between NaCl and non-Na+ stimuli. The third specific aim, which will be done in a collaborative effort with Dr. Timothy Gilbertson, uses perforated patch recording from hamster taste receptor cells maintained in an intact epithelium in order to determine (1) whether NaCl-responsive receptor cells also respond to non-Na+ salts and acids, (2) whether continual stimulation with NaCl leads to an adaptive decrease in taste receptor cell excitability that requires a similar time course to recover, and (3) whether adaptation to NaCl reduces the response to any other stimulus that activates the cell, i.e., that adaptation occurs at the whole-cell level. These findings will provide a basis for interpreting adaptation and cross-adaptation effects on hamster NST cells and in human psychophysical studies and will support the overall hypothesis that the cross-adaptation procedure reveals receptor commonalities.

DESCRIPTION (Adapted from investigator's abstract): Anatomical, electrophysiological and pharmacological data on gustatory and visceral pathways suggest several testable hypotheses about the functional organization of the nucleus of the solitary tract (NST). The PI's laboratory has shown that taste cells in the NST are driven by peripheral taste fibers via excitatory amino acids, that these cells are tonically inhibited by GABA and that taste-responsive cells in the NST are excited and sometimes inhibited by the neuropeptide, substance P. The current application will extend these findings by addressing three specific aims. First, the mechanisms of inhibition in the NST and its role in shaping the responsiveness of gustatory neurons will be further investigated. Initial experiments will determine if GABAB and glycine receptors contribute to inhibition in the NST and if inhibition sharpens the breadth of tuning of NST neurons to gustatory stimuli. Additional experiments will determine if there are differences in inhibitory control between gustator neurons that relay information to the parabrachial nuclei (relay neurons) and those that do not (nonrelay neurons). The second aim is to determine the influence of descending inputs from the gustatory neocortex and the central nucleus of the amygdala on NST taste neurons. Initial experiments will examine the hypothesis that descending inputs from the neocortex and amygdala produce excitatory and inhibitory modulation of NST cells. Subsequent experiments will test the hypotheses that the excitatory inputs are mediated by excitatory amin acids and the inhibitory inputs by inhibitory amino acids. The final set of experiments in this aim will evaluate the hypothesis that relay neurons are modulated differently by central inputs than nonrelay neurons. The final specific aim will address the question of modulation of NST taste responses by neuropeptides, and in particular, the apparent preferential excitation of NaCl-best neurons by substance P. The effects of met-enkephalin on NST taste neurons and the possible differential modulation of neuropeptide effects on relay and nonrelay neurons will also be examined. These in vivo experiments ar designed to reveal how the synaptic interactions in the NST and the connection to and from this nucleus control responses of NST neurons to gustatory inputs. These studies will provide important new information for our understanding of the functional organization of the central gustatory system.

Taste provides a model system for the study of regeneration because of the continual turnover of receptor cells and their trophic dependence on innervation by gustatory nerves. Studies on this project have shown that several cell-surface molecules are differentially expressed on taste cells and that this expression depends upon innervation by gustatory nerve fibers. The first Specific Aim is to determine the relationship between the expression of these molecules and taste cell type and whether this expression is determined by the innervating nerve or the gustatory epithelium. EM immunocytochemistry will address the relation between NCAM, L1, several blood group antigens, and other carbohydrates to taste cell type. The roles of the epithelium and the innervating nerve In determining the taste cell phenotype will be assessed by characterization of vallate and fungiform taste buds following cross-reinnervation of the posterior tongue by the chorda tympani (CT) nerve or the anterior tongue by the IXth nerve. The second Specific Aim is to determine the plasticity of central gustatory connections in the nucleus of the solitary tract (NST) following denervation and reinnervation of the taste buds. The first experiments will assess CNS responses to denervation. Preliminary studies show intense glial reactions in the NST following gustatory nerve injury as well as a significant constitutive expression of GAP-43 and astroglial and microglial markers in the NST. This suggests continual reorganization in the CNS accompanying normal taste cell turnover. The extent and time course of terminal degeneration and reactive gliosis following peripheral nerve injury will be studied by the cupric-silver degeneration method and antibodies against microglia (OX42) and astroglia (GFAP). Regeneration will be assessed using antibodies against regenerating axons (GAP43 and CDA-1). Golgi methods will be used to characterize the effects of taste nerve damage on NST cells. A second series of studies will examine the terminal field reorganization of regenerated IXth nerve axons following (a) IXth nerve crush, (b) IXth nerve crush combined with geniculate ganglion destruction, and (c) cross- anastomosis of proximal IXth to distal CT. Regenerated fibers will be labeled with HRP and their terminal fields mapped. Functional studies will localize responsive NST cells following the above three manipulations by mapping c-fos protein expression in response to electrical stimulation of the IXth nerve and by single-cell neurophysiological recording from the NST in response to gustatory, tactile, and thermal stimulation of the tongue.

taste buds; phenotype; taste; cell type; voltage /patch clamp; dyes; genetically modified animals; laboratory mouse;