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Schannen, Andrew

Biology

2005

MS

Organisms use olfaction to sense the chemical stimuli in their environment. Odorants are perceived through specific receptors, located on the cilia of olfactory sensory neurons. Through the olfactory signal transduction pathway organisms convert odorant cues into either inhibitory or excitatory stimuli, leading to different behaviors. Intracellular chloride is vital in determining whether an olfactory neuron exhibits an excitatory or inhibitory response to an odorant. Populations of isolated mouse OSNs have been shown to express a wide range intracellular Cl- [Cl]i resulting in varied responses to stimulus. To determine the mechanism of this variance, we examined two chloride cotransporters, NKCC1 and KCC2. NKCC1 moves one Na ion, one K ion and 2 Cl- ions into the cell while, KCC2 moves 1 K+ and 1 Cl- ion out of the cell. Since these are cotransporters, if anyone of ions being moved across the membrane is not present, no movement occurs. Western blots of nasal epithelium and immunocytohemistry of sectioned tissues, suggested that both KCC2 and NKCC1 were present in olfactory epithelium and that both transporters appeared to be located in the apical region of OSNs. To further characterize the expression patterns of KCC2 and NKCC1, isolated cells were examined using deconvolution microscopy. Both KCC2 and NKCC1 show intense staining in the dendritic knob and cilia of olfactory neurons. To show the physiological relevance of KCC2 and NKCC1 in mudpuppy olfactory neurons, chloride imaging of isolated OSNs was used. Substitution of choline for some of the bath sodium decreased the driving force for NKCC1 and thus it's function. Furosemide, a pharmacological inhibitor of KCC2 was used in the bath solution to determine the functionality of KCC2 in OSNs. Acetazolamide, an inhibitor of carbonic anhydrase modified our responses, suggesting involvement of a bicarbonate dependant mechanism for chloride accumulation. My work suggests NKCC1 and KCC2 are functional in olfactory neurons. In addition, my results suggest that another chloride transporter, most probably a chloride bicarbonate exchanger, is also functional in olfactory neurons and is another chloride transport mechanism involved in chloride homeostasis of OSNs.