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 individual-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.

SUMMARY Females may display dramatically different behavior depending on their state of ovulation. This is thought to occur through sex-specific hormones acting on behavioral centers in the brain. Whether incoming sensory activity also differs across the ovulation cycle to alter behavior has not been investigated. Here, we show that female mouse vomeronasal sensory neurons (VSNs) are temporarily and specifically rendered “blind” to a subset of male-emitted pheromone ligands during diestrus yet fully detect and respond to the same ligands during estrus. VSN silencing occurs through the action of the female sex-steroid progesterone. Not all VSNs are targeted for silencing; those detecting cat ligands remain continuously active irrespective of the estrous state. We identify the signaling components that account for the capacity of progesterone to target specific subsets of male-pheromone responsive neurons for inactivation. These findings indicate that internal physiology can selectively and directly modulate sensory input to produce state-specific behavior.

A variety of social behaviors like intermale aggression, fear, and mating rituals are important for sustenance of a species. In mice, these behaviors have been implicated to be mediated by peptide pheromones that are sensed by a class of G protein-coupled receptors, vomeronasal receptor type 2 (V2Rs), expressed in the pheromone detecting vomeronasal organ. Matching V2Rs with their cognate ligands is required to learn what receptors the biologically relevant pheromones are acting on. However, this feat has been greatly limited by the unavailability of appropriate heterologous tools commonly used to study ligand receptor specificity, because this family of receptors fails to traffic to the surface of heterologous cells. Here we show that calreticulin, a housekeeping chaperone commonly expressed in most eukaryotic cells, is sparsely expressed in the vomeronasal sensory neurons (VSNs). Correspondingly, knockdown of calreticulin in commonly available cell lines enables V2Rs to efficiently target to the cell membrane. Using this knowledge, we have now been able to successfully surface express receptors and functionally identify cognate ligands. Additionally, calreticulin4, a homolog of calreticulin shows restricted and enriched expression in the VSNs. Interestingly, in heterologous cells, calreticulin4 does not inhibit surface expression of V2Rs and can in part carry out functions of calreticulin. On the basis of our data, we postulate that V2Rs may use a unique trafficking mechanism whereby an important and more commonly expressed chaperone is deleterious for membrane export and is replaced by a functionally equivalent homolog that does not inhibit export while carrying out its functions.

Circadian rhythms synchronize physiological processes with the light-dark cycle and are regulated by a hierarchical system initiated in the suprachiasmatic nucleus, a hypothalamic region that receives direct photic input. The suprachiasmatic nucleus then entrains additional oscillators in the periphery. Circadian rhythms are maintained by a molecular transcriptional feedback loop, of which brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a key member. Disruption of circadian rhythms by deletion of the BMAL1 gene (Bmal1 knockout [KO]) induces a variety of disease states, including infertility in males, due to unidentified mechanisms. We find that, despite normal sperm function, Bmal1 KO males fail to mate with receptive females, indicating a behavioral defect. Mating is dependent on pheromone detection, as are several other behaviors. We determined that Bmal1 KO males also fail to display aggression and avoidance of predator scent, despite intact main olfactory function. Moreover, the vomeronasal organ, a specialized pheromone-responsive organ, was also functionally intact, as determined by calcium imaging in response to urine pheromone stimulus. However, neural circuit tracing using c-FOS activation revealed that, although Bmal1 KO males displayed appropriate activation in the olfactory bulb and accessory olfactory bulb, the bed nucleus of the stria terminalis and the medial preoptic area (areas responsible for integration of copulatory behaviors) failed to activate highly in response to the female scent. This indicates that neural signaling in select behavioral centers is impaired in the absence of BMAL1, likely underlying Bmal1 KO male copulatory defects, demonstrating the importance of the BMAL1 protein in the maintenance of neural circuits that drive pheromone-mediated mating behaviors.