Murine pheromone proteins constitute a context-dependent combinatorial code governing multiple social behaviors.

During social interactions, an individual's behavior is largely governed by the subset of signals emitted by others. Discrimination of "self" from "other" regulates the territorial urine countermarking behavior of mice. To identify the cues for this social discrimination and understand how they are interpreted, we designed an olfactory-dependent countermarking assay. We find major urinary proteins (MUPs) sufficient to elicit countermarking, and unlike other vomeronasal ligands that are detected by specifically tuned sensory neurons, MUPs are detected by a combinatorial strategy. A chemosensory signature of "self" that modulates behavior is developed via experience through exposure to a repertoire of MUPs. In contrast, aggression can be elicited by MUPs in an experience-independent but context-dependent manner. These findings reveal that individually emitted chemical cues can be interpreted based on their combinatorial permutation and relative ratios, and they can transmit both fixed and learned information to promote multiple behaviors.

Live cell calcium imaging of dissociated vomeronasal neurons.

Sensory neurons in the vomeronasal organ (VNO) are thought to mediate a specialized olfactory response. Currently, very little is known about the identity of stimulating ligands or their cognate receptors that initiate neural activation. Each sensory neuron is thought to express 1 of approximately 250 variants of Vmn1Rs, Vmn2Rs (A, B, or D), or FPRs which enables it to be tuned to a subset of ligands (Touhara and Vosshall, Annu Rev Physiol 71:307-332, 2009). The logic of how different sources of native odors or purified ligands are detected by this complex sensory repertoire remains mostly unknown. Here, we describe a method to compare and analyze the response of VNO sensory neurons to multiple stimuli using conventional calcium imaging. This method differs from other olfactory imaging approaches in that we dissociate the tightly packed sensory epithelium into individual single cells. The advantages of this approach include (1) the use of a relatively simple approach and inexpensive microscopy, (2) comparative analysis of several hundreds of neurons to multiple stimuli with single-cell resolution, and (3) the possibility of isolating single cells of interest to further analyze by molecular biology techniques including in situ RNA hybridization, immunofluorescence, or creating single-cell cDNA libraries (Malnic et al., Cell 96:713-723, 1999).

Synchronous evolution of an odor biosynthesis pathway and behavioral response.

BACKGROUND: Rodents use olfactory cues for species-specific behaviors. For example, mice emit odors to attract mates of the same species, but not competitors of closely related species. This implies rapid evolution of olfactory signaling, although odors and chemosensory receptors involved are unknown. RESULTS: Here, we identify a mouse chemosignal, trimethylamine, and its olfactory receptor, trace amine-associated receptor 5 (TAAR5), to be involved in species-specific social communication. Abundant (>1,000-fold increased) and sex-dependent trimethylamine production arose de novo along the Mus lineage after divergence from Mus caroli. The two-step trimethylamine biosynthesis pathway involves synergy between commensal microflora and a sex-dependent liver enzyme, flavin-containing monooxygenase 3 (FMO3), which oxidizes trimethylamine. One key evolutionary alteration in this pathway is the recent acquisition in Mus of male-specific Fmo3 gene repression. Coincident with its evolving biosynthesis, trimethylamine evokes species-specific behaviors, attracting mice, but repelling rats. Attraction to trimethylamine is abolished in TAAR5 knockout mice, and furthermore, attraction to mouse scent is impaired by enzymatic depletion of trimethylamine or TAAR5 knockout. CONCLUSIONS: TAAR5 is an evolutionarily conserved olfactory receptor required for a species-specific behavior. Synchronized changes in odor biosynthesis pathways and odor-evoked behaviors could ensure species-appropriate social interactions.