Functional analysis of human olfactory receptors with a high basal activity using LNCaP cell line.

Humans use a family of more than 400 olfactory receptors (ORs) to detect odorants. However, deorphanization of ORs is a critical issue because the functional properties of more than 80% of ORs remain unknown, thus, hampering our understanding of the relationship between receptor function and perception. HEK293 cells are the most commonly used heterologous expression system to determine the function of a given OR; however, they cannot functionally express a majority of ORs probably due to a lack of factor(s) required in cells in which ORs function endogenously. Interestingly, ORs have been known to be expressed in a variety of cells outside the nose and play critical physiological roles. These findings prompted us to test the capacity of cells to functionally express a specific repertoire of ORs. In this study, we selected three cell lines that endogenously express functional ORs. We demonstrated that human prostate carcinoma (LNCaP) cell lines successfully identified novel ligands for ORs that were not recognized when expressed in HEK293 cells. Further experiments suggested that the LNCaP cell line was effective for functional expression of ORs, especially with a high basal activity, which impeded the sensitive detection of ligand-mediated activity of ORs. This report provides an efficient functional assay system for a specific repertoire of ORs that cannot be characterized in current cell systems.

Transgenic labeling of higher order neuronal circuits linked to phospholipase C-ß2-expressing taste bud cells in medaka fish.

The sense of taste plays a pivotal role in the food-selecting behaviors of vertebrates. We have shown that the fish ortholog of the phospholipase C gene (plc-ß2) is expressed in a subpopulation of taste bud cells that transmit taste stimuli to the central nervous system to evoke favorable and aversive behaviors. We generated transgenic medaka expressing wheat germ agglutinin (WGA) under the control of a regulatory region of the medaka plc-ß2 gene to analyze the neuronal circuit connected to these sensory cells. Immunohistochemical analysis of the transgenic fish 12 days post fertilization revealed that the WGA protein was transferred to cranial sensory ganglia and several nuclei in the hindbrain. WGA signals were also detected in the secondary gustatory nucleus in the hindbrain of 3-month-old transgenic fish. WGA signals were observed in several diencephalic and telencephalic regions in 9-month-old transgenic fish. The age-dependent increase in the labeled brain regions strongly suggests that labeling occurred at taste bud cells and progressively extended to cranial nerves and neurons in the central nervous system. These data are the first to demonstrate the tracing of higher order gustatory neuronal circuitry that is associated with a specific subpopulation of taste bud cells. These results provide insight into the basic neuronal architecture of gustatory information processing that is common among vertebrates.