Molecular and cellular analysis of accessory olfactory circuits in mice

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In mice, the vomeronasal organ (VNO) is a main sensory organ for detecting pheromones, chemicals that affect social behaviors including: territoriality, sexual recognition and maternal care. The VNO can be divided into apical and basal layers that differ in their properties. VNO sensory neurons (VSNs) in the apical layer express members of the Vmn1r family of G protein coupled receptors (GPCRs), while basal VSNs express members of the Vmn2r family of GPCRs. Vmn2rs belong to the same family of GPCRs that includes the metabotropic glutamate, GABAB, and taste receptors. Basal VSNs do not obey the one receptor per neuron rule that is operative in all other sensory neurons in the olfactory system, with the exception of neurons of the newly identified necklace subsystem that do not express GPCRs. The Vmn2r family consists of four classes designated A, B, C and D. Members of classes A, B and D are more closely related to one another than to the seven members of class C (Vmn2r1-7). Each basal VSN expresses one Vmn2rC and one Vmn2rABD. Consequently, The seven class C receptors are broadly expressed in the VNO while class ABD receptors are sparsely expressed, like the rest of the olfactory receptors. The functional significance of the coexpression of Vmn2rs is not well understood. Based on our knowledge regarding the other members of the GPCR family to which Vmn2rs belong, we hypothesize that Vmn2rCs and Vmn2rABDs form heterodimers and that this interaction alters the functional properties of the basal VSNs. Vmn2rCs might affect the subcellular localization of Vmn2rABDs, their ligand binding and signaling properties. To examine these hypotheses, we have devised a multipronged strategy encompassing molecular and biochemical studies, neuroanatomical examination, and behavioral analysis. We have generated a mouse line carrying a deletion of the Vmn2r1-7 gene cluster using CRISPR/Cas9-mediated chromosome engineering followed by Cre recombination. Further, we generated a battery of specific antibodies against all Vmn2rCs, against specific class C receptors, and against a clade of Vmn2rAs. With these new reagents, we are poised to address these hypotheses. Our studies will reveal the mechanisms underlying the function of basal VSNs. More broadly, these experiments will shed light on how the olfactory system mediates social behaviors that are critical for the survival of the species. Thus far, only a handful of Vmn2r ligands have been identified. Since we predict that the deletion of the Vmn2rC cluster will affect the ability of the animals to properly respond to Vmn2rABD ligands, the cluster knockout line that we have generated will be invaluable for evaluating new Vmn2r ligands and their behavioral importance, as these ligands are identified. In this manner, our studies will deepen our understanding of the control of social behavior in multiple ways. Finally, our experiments will provide additional insight into the molecular mechanisms underlying the function of the GPCR family to which Vmn2rs belong. Some of these receptors have relevance to human disease.