VOLTAGE DYE CONFOCAL ANALYSIS OF OLFACTORY TRANSDUCTION

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Investigations of the odor-response properties of olfactory receptor neurons (ORNs) are difficult because a microelectrode records from one cell at a time and it takes an extended period of time to present each cell with many odors at several different concentrations. As a result it is not known how the nose distinguishes odor quality and intensity. This proposal uses confocal optical technology to image each of many ORNs in the living sensory epithelium simultaneously. The cell membranes are stained with voltage-sensitive dyes (VSDs) which change in fluorescence intensity when an odor stimulates the cells. Challenging technical problems including detection of small intensity changes, elimination of movement of the sensory organ, resolution of individual cells and of cell components such as cilia and internal organelles, minimizing effects of fading and toxicity due to light exposure, and repeatable odor delivery have all been resolved in this laboratory during the past three years. The experiments proposed are possible because of the laser scanning confocal microscope. This instrument produces an image of a very thin optical slice through thick tissues. Light from out-of-focus tissue above and below the section, which completely obscures cell boundaries and ultrastructure in the conventional epifluorescence microscope, is rejected in the confocal microscope. Experiments will determine which ORNs respond to each of many odors presented at low and high concentrations. The resulting response array will describe odor response selectivity, cell-to-cell similarity, response repeatability, and inactivation and recovery kinetics. Of particular importance is a test of the hypothesis that ORNs are highly selective, i.e., show high sensitivity to only one or a few odors. If so, presentation of an odor at low concentrations should evoke responses in only a few ORNs in the visual field of many hundred. The fraction of responding cells per odor in the epithelium will allow an estimate of the number of different stimulus substances likely to be required to characterize the ORN population. Differences in topographical organization of responding cells will determine the extent of anatomical clustering of similarily responding cells. Olfactory epithelia of frogs and salamanders will be compared because data obtained in the past suggests that these species are different with respect to odor transduction and chemotopic organization. Responses will be measured with normal mucus present and with mucus depleted to test the hypothesis that odor transduction requires an extracellular co-factor. VSDs have been identified which selectively stain supporting cells (SCs) in the olfactory epithelium. These cells are postulated to influence olfactory transduction. The extent and selectivity of odor-evoked membrane potential changes in SCs will be determined to test this postulate. VSDs and confocal microscopy are uniquely capable of resolving major issues in odor detection and coding which are not amenable to experimental study with microelectrode physiology. The olfactory sense has a major role in regulating body hormonal state, emotional disposition, hunger, and social behavior. The work proposed here will advance understanding of the cellular processes which subserve olfactory sensory function.