Characterization of olfactory receptor gene expression in the olfactory epithelium of larval Xenopus laevis

Abstract

Olfactory receptors comprise the largest multigene family of G protein-coupled receptors in organisms ranging from fish to primates and are the detectors of thousands of odorant molecules. As they are crucial elements for odor recognition, much effort was put into the analysis of how olfactory receptor neurons express olfactory receptor genes and how odorant information is encoded by the receptor proteins. It is nowadays expected that each olfactory receptor neuron expresses only a single odorant receptor gene and that each receptor can be activated by multiple stimuli. According to this, a combinatorial code for odor detection was proposed to explain peripheral coding of odorant stimuli in the olfactory system. However, some mostly recent studies point towards a revision of this scheme, especially with regard to the one receptor-one neuron hypothesis. Consequently, this thesis aims to investigate this discrepancy. In order to answer whether a single or multiple olfactory receptors are expressed in single olfactory receptor neurons of Xenopus larvae, a single cell reverse transcription PCR protocol was developed. The obtained data propose, that, at least during the developmental phase of Xenopus, a subset of olfactory receptor neurons exists exhibiting oligogenic expression of olfactory receptors. In addition to this straight expression assay, ligand-sensitivities of olfactory receptors were characterized. In order to retrospectively identify receptors that recognize a particular odorant of interest, an assay was established that combined Ca2+ imaging and single cell reverse transcription PCR. Expressed transcripts of olfactory receptor genes were thereby amplified from those olfactory neurons that showed odorant responsiveness to certain amino acids. This functional strategy led to the finding of one candidate receptor that may be sensitive to these stimuli.