Enhancement, Modulation and Electrophysiological Characterization of Murine Olfactory Neurons to Odorant Stimulation in Vitro
Type of DegreeDissertation
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The objectives of this study is to electrophysiologically characterize cultured olfactory epithelium (OE) and to compare with that of rapidly isolated OE; to establish the Gai involvement in the modulation of adenylyl cyclase (AC) and to demonstrate the participation of zinc in odorant stimulated enhancement of smell. The murine family has a well developed sense of smell and is widely used in the field of olfactory research. Producing a long lived, readily accessible, economically feasible and physiologically viable cultured OE model would be ideal, to meet with specific requirements and nature of the research. The cultured OE of different ages were exposed to charcoal-filtered air, individual odorants, and an odorant mixture of (+)carvone, (-)carvone, eugenol, and ethyl butyrate at vi varying concentrations or with varying inter pulse periods to explore the system’s ability to respond to and adapt to odorant presentation. The study also involved with Gai-Protein Inhibition of olfactory signal transduction. The whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous Gi protein negatively regulates odorant-evoked intracellular signaling. Rat OE were subjected to whole cell recordings with holding membrane potential of +/- 57 mV. The patch electrode (resistance of 8±3MO) was filled with intracellular fluid (ICF) in the presence or absence of antibodies directed against the Gas/olf-subunits (Santa Cruz Biotech.), antibodies raised against Gai-subunits (Calbiochem) or control antibodies. In addition to G proteins involvement in the enhancement, modulation and inhibition of Odorant signals, there is evidence that metals like zinc enhances odorant induced response of Rat OE are exposed to crystalline metallic nanoparticles which are found in proteon nucleating centers (PNC) and metallic Zinc nanoparticles (artificial PNC). EOG recordings were made from dissected rat olfactory receptor neurons under control conditions (in the presence of extra cellular fluid -ECF) and experimental conditions (in the presence of PNC and Zn and Cu).The metal nano particles were integrated with the odorants of interest and were delivered onto the odor receptors. Our results show that in cultured tissues the characteristics of the EOG responses are similar to that of standard EOG responses of dissected OE. Cultures one week old or less (n = 8) showed rapid rise times but prolonged decay times. Cultures aged 13 to 15 days in vitro (n = 8) showed both rapid rise and decay times. Although all cultures responded to the four individual odorants in equimolar concentrations, their response to vii each odorant was variable; ethyl butyrate elicited the strongest response on average. High frequency stimulation with inter pulse intervals of 200 ms, the EOG responses appeared to be a summative effect due to closely spaced inter stimuli intervals. At longer durations of the inter-stimuli intervals (800 ms), adaptation was clearly apparent with the second and succeeding responses of lower amplitude than the initial response. This trend continued until the duration of the inter-stimuli intervals reached 20 seconds, where the second and succeeding response amplitudes were equal to that of the first EOG amplitude. These results extend the characterization of our olfactory epithelium-olfactory bulb organotypic culture system and suggest that olfactory epithelium passes through an immature stage in the week following explanation and culturing. The rapidly isolated rat OE with intracellular microinjections Gas/olf antibodies inhibited current responses elicited by odorants. In contrast, antibodies raised against Gaisubunits caused a strong enhancement of the odorant-induced currents. When IgG or antiolfactory marker protein antibody was substituted for G-protein antibodies, the odorantinduced currents were not significantly changed.