A genetic platform for functionally profiling odorant receptors in olfactory cilia ex vivo

Abstract

The molecular basis for odor perception in humans remains enigmatic because of the difficulty in studying odorant receptors (ORs) outside their native environment. Efforts toward OR expression and functional profiling have been met with limited success because of the poor efficiency of their cell surface expression in vitro. Structures protruding from the surface of olfactory sensory neurons called cilia contain all of the components of the olfactory signal transduction machinery and can be placed in an ex vivo plate assay to rapidly measure odor-specific responses. Here, we describe an approach using cilia isolated from the olfactory sensory neurons of mice expressing two human ORs, OR1A1 and OR5AN1, that showed 10- to 100-fold more sensitivity to ligands as compared to previous assays. A single mouse can produce enough olfactory cilia for up to 4000 384-well assay wells, and isolated cilia can be stored frozen and thus preserved. This pipeline offers a sensitive and highly scalable ex vivo odor-screening platform that has the potential to decode human olfaction.

Technique of flat-mount immunostaining for mapping the olfactory epithelium and counting the olfactory sensory neurons

“…A list of publications in which immunostaining on flatmounted septal OE was developed and/or used is summarized in Table 2 [9,11,12,[23][24][25][26][27][28][29][30][31][32]. The other en face imaging methods, such as fluorescent protein transgenic animals [33][34][35] or dolichos biflorus agglutinin (DBA) staining [36,37] were not included in the table because of their technical differences. The rare use of the technique of immunostaining on flat OE is probably due to technical pitfalls.…”