Author Correction: Female mouse tears contain an anti-aggression pheromone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Female mouse tears contain an anti-aggression pheromone.

Tears contain pheromones that trigger specific behavioral responses. In the mouse, male tear fluid is involved in long and short-term effects such as the receptive behavior and pregnancy block in females and the aggression in males. In contrast, pup tears exert an inhibitory effect on male mating behavior, also promoting sexual rejection in females. In the rat, a male lacrimal protein acts as an intraspecific and heterospecific signal enhancing sexual behavior in females and evoking avoidance behavior in mouse. However, behavioral effects of female tears on male behavior have yet to be described. Here, we report that female lacrimal fluid of different mouse strains contains a relatively small and involatile factor that abolishes inter-male aggression switching it into a copulatory behavior. The production of this molecule by the lacrimal glands is not affected by the estrous cycle but it is sensitive to ovariectomy, thus suggesting a control mediated by hormones. Moreover, this lacrimal anti-aggression pheromone modulates the activity of the lateral habenula, a brain area responsible for the valence of the aggressive interactions.

In-vivo activation of vomeronasal neurons shows adaptive responses to pheromonal stimuli.

In most mammals, the vomeronasal system has a pivotal role in mediating socio-sexual behaviours. The vomeronasal organ senses pheromones through the activation of specific receptors. Pheromone binding to cognate receptors activates Ca-influx via the gating of a cation channel that generates membrane depolarisation. The ex-vivo activation of vomeronasal neurons (VSNs) by pheromonal stimuli has been largely investigated by electrophysiological and imaging techniques; however, few studies have been carried out to determine the physiological responses of VSNs, in-vivo. By tracking the phosphorylation of S6 ribosomal protein as a marker of neuronal activity, we show that S6 becomes phosphorylated (pS6) in mouse VSNs stimulated by intraspecific and heterospecific pheromonal cues. We observed that female scent induces pS6 immunoreactivity in the apical VSNs of male vomeronasal epithelium, whereas male cues stimulate S6 phosphorylation in both the basal and apical VSNs of females. We also show that this dimorphic pattern of pS6 immunoreactivity is reproduced when heterospecific stimuli are used. Moreover, we found that a consistent proportion of VSNs is activated by both heterospecific and intraspecific pheromones. Additionally, we have evidence of adaptive responses to S6 phosphorylation when stimulation with cues of the same and opposite sex and of different species is sustained.

Combinatorial co-expression of pheromone receptors, V2Rs.

Basal neurons of the vomeronasal organ of the mouse express a superfamily of about 120 pheromone receptors, named V2Rs, that are grouped in four families, A, B, C, and D, according to sequence homology. Family-A, -B, and -D V2Rs are expressed as one receptor gene per cell, but we previously reported their co-expression with family-C V2Rs. Here, we show that basal neurons can be further grouped according to the combinatorial expression of different V2Rs. Altogether, these findings suggest that in each basal neuron a transcriptional program is active for expressing a combination of two compatible receptors and for excluding, at the same time, the expression of all other V2Rs. Further analyses revealed non-random combinations of co-expression between family-C V2Rs and genes of the class Ib major histocompatibility complex. Thus, each basal neuron of the vomeronasal organ represents a highly qualified sensory unit for detecting very specific combinations of pheromonal cues.

Evolution of spatially coexpressed families of type-2 vomeronasal receptors in rodents.

The vomeronasal organ (VNO) is an olfactory structure for the detection of pheromones. VNO neurons express three groups of unrelated G-protein-coupled receptors. Type-2 vomeronasal receptors (V2Rs) are specifically localized in the basal neurons of the VNO and are believed to sense protein pheromones eliciting specific reproductive behaviors. In murine species, V2Rs are organized into four families. Family-ABD V2Rs are expressed monogenically and coexpress with family-C V2Rs of either subfamily C1 (V2RC1) or subfamily C2 (V2RC2), according to a coordinate temporal diagram. Neurons expressing the phylogenetically ancient V2RC1 coexpress family-BD V2Rs or a specific group of subfamily-A V2Rs (V2RA8-10), whereas a second neuronal subset (V2RC2-positive) coexpresses a recently expanded group of five subfamily-A V2Rs (V2RA1-5) along with vomeronasal-specific Major Histocompatibility Complex molecules (H2-Mv). Through database mining and Sanger sequencing, we have analyzed the onset, diversification, and expansion of the V2R-families throughout the phylogeny of Rodentia. Our results suggest that the separation of V2RC1 and V2RC2 occurred in a Cricetidae ancestor in coincidence with the evolution of the H2-Mv genes; this phylogenetic event did not correspond with the origin of the coexpressing V2RA1-5 genes, which dates back to an ancestral myomorphan lineage. Interestingly, the evolution of receptors within the V2RA1-5 group may be implicated in the origin and diversification of some of the V2R putative cognate ligands, the exocrine secreting peptides. The establishment of V2RC2, which probably reflects the complex expansion and diversification of family-A V2Rs, generated receptors that have probably acquired a more subtle functional specificity.

A recent class of chemosensory neurons developed in mouse and rat.

In most animal species, the vomeronasal organ ensures the individual recognition of conspecifics, a prerequisite for a successful reproduction. The vomeronasal organ expresses several receptors for pheromone detection. Mouse vomeronasal type-2 receptors (V2Rs) are restricted to the basal neurons of this organ and organized in four families. Family-A, B and D (family ABD) V2Rs are expressed monogenically (one receptor per neuron) and coexpress with either Vmn2r1 or Vmn2r2, two members of family-C V2Rs. Thus, basal neurons are characterized by specific combinations of two V2Rs. To investigate this issue, we raised antibodies against all family-C V2Rs and analyzed their expression pattern. We found that six out of seven family-C V2Rs (Vmn2r2-7) largely coexpressed and that none of the anti-Vmn2r2-7 antibodies significantly stained Vmn2r1 positive neurons. Thus, basal neurons are divided into two complementary subsets. The first subset (Vmn2r1-positive) preferentially coexpresses a distinct group of family-ABD V2Rs, whereas the second subset (Vmn2r2-7-positive) coexpresses the remaining group of V2Rs. Phylogenetic reconstruction and the analysis of genetic loci in various species reveal that receptors expressed by this second neuronal subset are recent branches of the V2R tree exclusively present in mouse and rat. Conversely, V2Rs expressed in Vmn2r1 positive neurons, are phylogenetically ancient and found in most vertebrates including rodents. Noticeably, the more recent neuronal subset expresses a type of Major Histocompatibility Complex genes only found in murine species. These results indicate that the expansion of the V2R repertoire in a murine ancestor occurred with the establishment of a new population of vomeronasal neurons in which coexists the polygenic expression of a recent group of family-C V2Rs (Vmn2r2-7) and the monogenic expression of a recent group of family-ABD V2Rs. This evolutionary innovation could provide a molecular rationale for the exquisite ability in individual recognition and mate choice of murine species.

The vomeronasal receptor V2R2 does not require escort molecules for expression in heterologous systems.

In rodents, many behavioural responses are triggered by pheromones. These molecules are believed to bind and activate two families of G-protein coupled receptors, namely V1Rs and V2Rs, which are specifically expressed in the chemosensory neurons of the vomeronasal organ. V2Rs are homologous with Group 3 of G-protein-coupled receptors, which includes metabotropic glutamate receptors, calcium-sensing receptors, fish olfactory receptors, and taste receptors for sweet molecules and amino acids. The large extracellular region of these receptors is folded as a dimer and, in this form, binds agonists that in many cases are amino acids. It has recently been reported that V2Rs must be physically associated with specific major histocompatibility complex class Ib molecules (MHC) for their expression in both mouse vomeronasal neurons and heterologous cell lines. Here, we show that in contrast to the other V2Rs, V2R2, an atypical member of this receptor family, can be successfully and abundantly expressed by insect cells without the requirement of escort molecules like MHC. Moreover, the extracellular binding domain of V2R2, secreted as a soluble product, forms dimers via cysteine-mediated sulphur bridges. Overall, the data presented in this paper confirm that V2R2 diverges from the other members of the V2R family and suggest a different role for this receptor in pheromonal communication.