To make sense of the complex chemical world, our sense of smell encodes olfactory stimuli combinatorially, in which different odors are recognized by distinct combinations of odor receptors (OR). This lets us detect of a vast range of smells with a limited number of ORs. Despite the importance of smell in guiding critical decisions such as what food to eat or what mate to select, we still have a poor understanding of the neural mechanisms behind combinatorial olfactory sensing. To fill this gap in knowledge, Timothy Holy, PhD, the Alan A. and Edith L. Wolff Professor of Neuroscience at Washington University School of Medicine, will study how the olfactory system encodes chemosensory information with support from a $2.1 million R01 grant awarded by the National Institutes of Health.
In the mouse, there are four recognized olfactory subsystems: the main olfactory system (MOS), septal organ, Gruneberg ganglion and vomeronasal organ (VNO). The Holy Lab will study combinatorial coding in the vomeronasal system, which is responsible for sensing pheromones. The VNO offers several advantages to researchers. Unlike the MOS where the volatiles that it detects can excite dozens to hundreds of different receptor types, individual non-volatile compounds typically activate fewer than 10 vomeronasal receptor (VR) types. And, while volatile compounds detected by the MOS are “floppy,” having rotatable bonds that allow them to adopt a large number of different structural conformations, VR ligands are rigid in structure, which makes structure-activity analyses simpler. The activity patterns of every VR type in response to a chosen set of ligands can be exhaustively recorded in the mouse VNO by massively-parallel calcium imaging via light sheet microscopy (LSM), a technique that was first accomplished in the Holy Lab.
To study the combinatorial code of the vomeronasal systems, the Holy Lab will look at the structural features of ligands and receptors that influence their interactions. First, they will screen a large panel of ligands against the entire VR population in the mouse VNO and monitor the response of each neuron via LSM. The Holy Lab will then perform computational structure modeling to analyze the commonalities and differences between ligands that share similar LSM profiles to identify their pharmacophores, which are structural features of the ligands that contribute to their receptor binding.
This research has the potential to reveal new principles that will change our understanding of the evolutionary forces that shape olfaction.
Tim Holy, PhD
Another important step in developing their pharmacophore model is matching VRs with the pharmacophores they respond to. To do this, they will visualize pairings of ligands with the VRs they activate using a technique the Holy Lab developed called physiological optical tagging sequencing (PhOTseq) in which activated neurons are tagged long-term with a photoactivatable fluorescence protein. Tagged neurons are isolated by fluorescence-activated cell sorting and subjected to expression profiling via RNA-seq to identify the VR that these cells express (each sensory neuron typically only expresses one receptor gene at a high level). By characterizing ligand structures and determining which pharmacophores are sensed by which VRs, the researchers will develop a quantitative pharmacophore model that allows for the prediction of responses to novel ligands.
For the second arm of this project, they will link receptor sequence and function. To do this, they will individually express 37 homologous VRs in the VNO of different animals and measure their response to a panel of ligands via LSM. Through functional comparisons and analyses of sequence homology, they will identify candidate amino acid residues in VR receptors that are important for chemosensation and validate them via site mutagenesis.
After characterizing pharmacophores and the chosen VRs, the researchers will link ligand and receptor features that are important for binding to develop a system-wide framework for predicting and manipulating ligand-receptor interactions. According to Holy, this research has “the potential to reveal new principles that will change our understanding of the evolutionary forces that shape olfaction.”