We have recently developed a genetic, customizable platform, called MouSensor, that can be used to express -for the first time- any odorant receptor protein (OR), including human ORs, in a large population of mouse olfactory sensory neurons, providing a breakthrough technology with a wide array of translational applications (D'Hulst et al., 2016). ORs expressed through this platform technology are fully functional and retain the one-receptor-per-neuron expression pattern. Moreover, behavioral tests show that our transgenic mice have an increased sensitivity for the specific odor that activates the cloned receptor. We ultimately envision transforming our mouse OR bioreactors into a bio-nose-on-a-chip by combining extracted MouSensor neurons with chip technology. The first technical hurdle we have to overcome in achieving this goal is to develop a reliable and quantitative objective analysis of OR activation ex vivo. In the olfactory system, when an odor activates an OR, a G protein cascade gets activated whereby cAMP levels rise dramatically leading to calcium entry through cyclic-nucleotide gated channels. Using genetically-encoded reporters for Calcium and/or cAMP is a feasible solution to report odor-evoked signaling. This entire cascade initiates in the cilia appendages attached to the dendritic knob; olfactory cilia can be readily detached from the neuron providing an ex vivo system to assess OR activation. The research proposed here allows us to develop an optical, objective read-out upon specific OR activation, by targeting genetically-encoded Calcium or cAMP reporters to olfactory cilia. Importantly, cilia have been shown to retain their activity after freezing, dramatically increasing the shelf-life of the assay. Here, we propose to target two ultrasensitive fluorescence- associated reporter proteins to primary cilia and provide a breakthrough technology to monitor Ca2+ and/or cAMP signaling in olfactory cilia by generating two different reporter mice. We will use CRISPR/cas9 genome editing to create fusion proteins to a cilia trafficking protein that we already know targets to cilia when fused to the Green Fluorescent Protein (GFP). Combining either of the new reporter lines with our established MouSensor technology will provide a streamlined system for high-throughput odor profiling and may lead to development of novel reporters for neuronal cilia signaling in general.
/Relevance Statement Neuronal signaling through cilia in the nervous system is a fundamental process of cell communication and, when defective, is a major cause of human diseases and developmental disorders, commonly referred to as ciliopathies. Imaging signaling directly at its source may reshape how we understand neuronal function.