The discovery of large receptor families mediating olfactory and vomeronasal chemosensation has offered a unique opportunity to decode the molecular logic by which environmental information influences animal behavior3,4
. The vomeronasal organ (VNO) of rodents plays a critical role in identifying sex-and species- specific chemical cues and in mediating mating, territorial aggression, defensive responses to predators and associated endocrine changes1,5
. With rare exceptions6,7,8
, the molecular identity of VNO receptors (VRs) recognizing distinct animal cues is unknown, thus limiting the ability to explore the sensory mechanisms underlying behavioral specificity. Prior studies suggested that vomeronasal detection is extremely sensitive and narrowly tuned to male, female, or heterospecific cues5,9,10,11
, but they have not allowed the identification of the activated receptors. We describe here a robust and high-throughput molecular readout of vomeronasal activation that enabled us to uncover the receptor specificity of 88 individual VRs to a vast range of ethologically relevant cues. These results establish the molecular and functional framework underlying vomeronasal signaling.
In initial experiments, we exposed female mice to male or clean bedding and assessed the upregulation of the immediate early genes (IEGs) Arc
, and Nr4a1
by in situ hybridization on VNO tissue. Our data show that the sensitivity of Egr1
induction following semiochemical exposure far exceeds that of other IEGs () (60.1 ± 7.1 cells per 0.2 mm2
, 7.9 ± 1.9 cells per 0.2 mm2
). Indeed c-Fos
, an IEG used in previous VNO stimulation studies labels only a subset of Egr1
positive cells (Supplementary fig. 1
). In TrpC2−/−
mutants, in which VNO activation is genetically impaired12
induction after semiochemical exposure is completely abolished (n=3), demonstrating the specificity of Egr1
activation following sensory stimulation ().
Egr1 expression is robustly induced by pheromone-evoked VNO neuronal activation
We then exposed animals to 29 distinct ethologically relevant cues5,13
. Male and female bedding from different mouse subspecies and wild-derived strains, as well as a variety of heterospecific cues from sympatric competitors and predators robustly induced Egr1
expression in the VNO (). Remarkably, food-related insect stimuli and cues from presumably neutral species such as woodchuck failed to generate VNO activation.
Widespread activation of VNO receptors by conspecific and heterospecific cues
V1R and V2R neurons were equally activated by a large variety of stimuli as judged by co-labeling of Egr1
, a marker of V1R-expressing neurons14,15
(, Supplementary fig. 2a
). Interestingly, simultaneous exposure to multiple cues from the same class of animals (e.g., Peromyscus
species, reptiles, or predatory birds) did not significantly increase the number of Egr1
+ cells when compared to activation by a single stimulus (p>0.4, two tailed t-test when the strongest of each stimulus class was compared to the corresponding mix), indicating that neuronal populations activated by related animals are largely overlapping (). In contrast, simultaneous exposure to all heterospecific stimuli significantly increased Egr1
+ cells from 5 to 10 % per cue to up to ~30 % (p<0.01, two tailed t-test), indicating that distinct heterospecific cues have different response profiles. Moreover, while mouse bedding activated 5 to 7 % of VNO neurons in animals of the opposite sex, mixes of conspecific and heterospecific scents activated ~35 % of neurons () suggesting that receptors activated by both types of cues are also largely distinct.
To assess Egr1
as readout of VNO activation, we compared it to cue-evoked neuronal responses visualized by the genetically encoded calcium indicator G-CaMP316
. Strikingly, Egr1
and G-CaMP3 reported remarkably similar patterns of activities in the basal, or basal plus apical VNO neuroepithelium following exposure to rat and snake stimuli, respectively (), confirming Egr1
induction as an exquisitely sensitive and accurate marker of VNO neuronal activation.
Next, we developed a high-throughput platform to uncover the receptors activated by specific cues. With the exception of widely expressed V2R2 receptors17
, vomeronasal neurons are thought to express a unique receptor gene from the V1Rs or V2Rs. We generated 209 RNA probes that specifically identify individual or subgroups of VRs by in situ hybridization. A collection of clade-specific probes was designed to target all receptor sequences within each of the 8 distinct V1R or V2R clades (). Probes with higher specificity that readily distinguish a single or few closely related VR sequences were designed based on divergent 5’UTR/intron18
and 3’UTR regions in VR genes. The specificity of these probes for closely related VRs was validated by dual color in situ hybridization (Supplementary fig. 3
). While detecting all VRs at single gene resolution was technically impossible, altogether our probes targeted 139 distinct VRs with specificity of a single or at most few genes.
We then used a hierarchical approach to systematically uncover VRs activated by distinct cues (Supplementary fig. 2b, 4
). First, the co-expression of Egr1
with either Gαi2
or formyl peptide receptors (FPRs)19,20
identified the nature of the activated neurons as expressing a V1R, V2R or FPR, respectively. Most stimuli activated both V1R- and V2R-expressing neurons, while few activated only V1R- (hawk and owls) or V2R-expressing cells (rat, fox and male mouse cues in females) (Supplementary table 1
). We found no activation of FPR-expressing cells. We then assessed the specific V1R or V2R clades associated with the activated neurons (, Supplementary fig. 2c
). Interestingly, some clades appeared specifically stimulated by a distinct class of cues, for example V1Rd and V2R clades 4 and 7 by sex-specific cues. Subsequently, receptor specific probes were used to unmask the exact molecular identity of the Egr1
+ cells. By collecting data from 9,948 VNO slices, each containing approximately 1000 neurons, we succeeded in the identification of 88 receptors (56 V1Rs and 32 V2Rs, 78 single and 10 unresolved receptors) associated with distinct cues (Supplementary fig. 5
, Supplementary table 1, 2
). Importantly, these receptors span most V1R and V2R clades, thus generating the most comprehensive functional map of vomeronasal receptors to date.
The vomeronasal system plays an essential role in regulating sex-specific behaviors. Previous reports suggest that vomeronasal neurons detect sex-specific cues in mouse urine, tear and saliva9,10,13,21,22
, and Vmn2r116 (or V2Rp5) was identified as detecting the male pheromone ESP16
(Supplementary fig. 6
). Our strategy uncovered 28 receptors (25 single, 3 unresolved) detecting mouse cues, among which 26 detecting sex-specific cues (, Supplementary table 1
). Only two receptors (V1ri9, V1ri10) responded to both male and female mouse cues, consistent with the desensitization of IEG induction in vivo by self-secreted stimuli6
. Four receptors (V1re2, V1re3, V1re6, V1rg6) were selectively activated by female cues in males and females, while a larger set of V1Rs and V2Rs responded to female cues only in males (, Supplementary table 1
). In addition, responses to male-specific signals involved Vmn2r116, Vmn2r28, Vmn2r15, Vmn2r16, and Vmn2r17 in males and females, Vmn2r66 and Vmn2r82 in females, and Vmn2r84/85/86/87 and Vmn2r88 in males (, Supplementary table 1
). Interestingly, no V1R was found to specifically respond to male cues. Thus, consistent with a previous report9
, the detection of sex-specific cues appears to rely on a small and specific subset of VNO neurons, the identity of which is now clearly established. This molecular logic is likely to underlie the initiation of sex-dependent behavioral interactions, such as male-male aggression and mating behaviors.
Receptor repertoires to sex-specific cues
Vomeronasal detection of heterospecific cues, or kairomones, is involved in the adaptive defensive behaviors5,13,23
. Indeed, rat bedding induces robust avoidance to the predator cues in TrpC2+/−
but not in TrpC2−/−
animals (). Moreover TrpC2−/−
animals exhibited abnormal ingestive behavior of the predator bedding suggesting that VNO inputs also inhibits foraging24,25
(Supplementary fig. 7
Receptor repertoires to heterospecific cues
We report here the identity of 71 (63 single, 8 unresolved) receptors activated by heterospecific scents. Consistent with the distinct behavioral outputs generated by pheromones and kairomones, we found that only 11 receptors were common to both types of cues, while 60 were uniquely activated by heterospecific stimuli, and 17 by mouse cues only (). The detection of kairomones thus emerges as a major function of the VNO5,13
. The identity of one of the identified receptor population for the detection of predator cues was confirmed independently by Egr1
activation in cells expressing YFP under the V1Rh7 promoter26
(Supplementary fig. 8
). Further, loose patch recording of V1Rh7-YFP expressing neurons demonstrated significant increase in firing rates following exposure to ferret, but not to rat stimuli (1.732±0.170 Hz for ferret, 0.420±0.061 Hz for rat, n = 4) (, Supplementary fig. 9
Remarkably, some receptors show unique association with distinct classes of predators. Vmn2r89 and Vmn2r121 were exclusively activated by scents from snakes, V1rc10/11/12 by owls. Also, up to 70 % of V2R clade 5 neurons were activated by every mammalian predator tested, but not by sympatric non-predators (, Supplementary fig. 5, 10
). Moreover, each predator cue generated a distinct receptor signature: for example, rat stimuli activate Vmn2r59, Vmn2r60, Vmn2r61, Vmn2r108, and Vmn2r110, all within clade 8, while ferret cues activate V1rf5 and Vmn2r77/78/79, suggesting that the mouse VNO has the sensory machinery to discriminate predator species.
We then searched for receptors detecting sympatric species Mus spicilegus
and Mus musculus
, which diverged evolutionarily ~1.5 million years ago and do not breed in the wild27,28
. Receptors activated by M. spicilegus
and M. musculus
male cues appear mostly distinct, though often closely related (Supplementary fig. 5, 11
). For example, Vmn2r8/9 and Vmn2r11, activated by M. spicilegus
, and Vmn2r15, Vmn2r16 and Vmn2r17, activated by M. musculus
, belong to clade 6 (Supplementary fig. 11b
). Likewise, Vmn2r69 activated by M. spicilegus
and Vmn2r66 by M. musculus
belong to clade 3. Thus, through the activation of specialized receptors, M. musculus
may readily discriminate scents emitted by closely related but reproductively incompatible species, a property that could be linked to the reproductive isolation of these species.
V1Rs and V2Rs are associated with segregated neural pathways29
, raising the possibility that fundamental functional differences may exist between the two families. Remarkably, our data suggest that V1Rs and V2Rs display different receptor properties. Nearly half of the V1Rs (27 out of 56) exhibit generalized activation by multiple cues (), including signals with apparent conflicting behavioral significance. For example, receptors within the V1Rh, V1Rc and V1Re clades were activated by mouse, predator and non-predator cues (Supplementary table 1, 2, Supplementary fig. 12
). In contrast, most V2Rs (29 out of 32) are activated by cues reflecting a unique ethological context such as a male, female, or a given type of predator or non-predator. In addition, hierarchical clustering across all identified receptors revealed clear segregation between V1Rs and V2Rs (Supplementary fig. 5
). These results suggest that V1R and V2R pathways may encode different types of information: individual V2Rs appear uniquely poised to encode information about the identity of emitters with clear behavioral significance, for example the sex of a conspecific or the predator or competitor nature of a heterospecific. In contrast, individual V1Rs may encode other forms of biologically relevant information.
To gain further insight into the molecular logic of V1R-mediated signaling, we investigated the detection of sulfated steroids, thought to account for 80 % of VNO neuronal activation by female urine30
likely through V1Rs11
. Our data show that, when male mice were exposed to a mix of synthetic steroid sulfates, receptors from V1Ref and V1Rjk clades were specifically activated (). We then tested individual compounds to attempt the pairing of specific steroid ligands with their cognate receptors. Corticosterone-21 sulfate (Q1570), a compound in female urine30
, strongly activated V1re2
and more weakly V1re6
cells (). Both receptors were shown in earlier experiments to be specifically activated by female cues (). In addition, we uncovered strong activation of V1rf3 by 17β-estradiol sulfate (E1050) and V1rj2 by both E1050 and 5-androstene-3β, 17β-diol disulfate (A7864) (), although these two receptors were not activated by female bedding, indicating that these steroids are not secreted under normal conditions.
Sulfated steroids detection by V1Rs
Thus, our approach efficiently achieved single compound resolution, offering the unique opportunity to test the receptor specificity to a variety of individual chemicals. We further tested 4 sulfated estrogen compounds structurally related to E1050, and 3 additional sulfated pregnenes structurally related to Q1570. V1rf3 appeared broadly selective to estradiols, estriols, and related stereoisomers, regardless of sulfate positions, but did not respond to androgens or glucocorticoids (). Interestingly, no other V1rf receptor was activated by these ligands. In contrast, V1rj2 was activated by androgens and estradiols but not estriols. Similarly, V1re2 and V1re6 selectively detected corticosteroids (). Therefore, V1R receptors can distinguish distinct structural classes of steroids. Androgens, estrogens, and glucocorticoids are ubiquitous though sensitive reporters of the animal endocrine state. Our results thus suggest that V1Rs may serve as detectors of the physiological status of an animal.
In conclusion, our data have begun to uncover the molecular logic by which vomeronasal receptors of different families, clades, and receptor sequences extract biological information and trigger appropriate behavioral responses to animals of a given sex, species and physiological status. The collection of receptors uncovered in this study provides a molecular foundation to further dissect the neural circuits governing social and sexual communication in rodents.