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Proc Natl Acad Sci U S A. Aug 2, 2011; 108(31): 12898–12903.
Published online Jul 18, 2011. doi:  10.1073/pnas.1107770108
PMCID: PMC3150917
Neuroscience

G protein Gαo is essential for vomeronasal function and aggressive behavior in mice

Abstract

The rodent vomeronasal organ (VNO) mediates the regulation of species-specific and interspecies social behaviors. We have used gene targeting to examine the role of the G protein Gαo, encoded by the gene Gnao1, in vomeronasal function. We used the Cre-loxP system to delete Gαo in those cells that express olfactory marker protein, which includes all vomeronasal sensory neurons of the basal layer of the VNO sensory epithelium. Using electrophysiology and calcium imaging, we show that the conditional null mice exhibit strikingly reduced sensory responses in V2R receptor-expressing vomeronasal sensory neurons to specific molecular cues, including MHC1 antigens, major urinary proteins, and exocrine gland-secreting peptide. Gαo is also vital for vomeronasal sensing of two N-formylated mitochondrially encoded peptides derived from NADH dehydrogenase 1. Furthermore, we show that Gαo is an essential requirement for the display of male–male territorial aggression as well as maternal aggression in mice. Finally, we show that Gαo-dependent maternal aggression can be induced by major urinary proteins. These cellular and behavioral phenotypes identify Gαo as the primary G-protein α-subunit mediating the detection of peptide and protein pheromones by sensory neurons of the VNO.

Keywords: formyl peptide receptor, olfaction, peptide detection, Trpc2, social recognition

The mammalian olfactory system comprises a complex array of subsystems, but the functional significance of this organization remains unclear (1). The mouse vomeronasal organ (VNO)—which has attracted specific attention because of its vital role in social communication (2, 3)—consists of at least two distinct layers containing apical (superficial) and basal (deep) populations of vomeronasal sensory neurons (VSNs), which project their axons to discrete regions of the accessory olfactory bulb (AOB) (46). These VSN populations are characterized by differential expression of heterotrimeric G proteins and G protein-coupled receptors (GPCRs): apical VSNs express Gαi2 and members of the V1R family of vomeronasal GPCRs (7), whereas basal VSNs express Gαo and members of the V2R receptor family (810). VSNs of both layers express the transient receptor potential channel Trpc2 (1113). Whether these two VNO subsystems function in relative isolation, each specialized to control a distinct subset of social behaviors, or whether they work in a cooperative and integrative manner remains unclear.

A series of gene deletion studies has revealed an obligate requirement of Trpc2 (1214) and at least some V1Rs (15, 16) and V2Rs (17, 18) for the generation of sensory responses in VSN populations. Together, these studies support a model of VSN signaling in which receptor occupancy, through G-protein coupling, causes activation of phospholipase C (PLC) that, in turn, leads to the activation of a Ca2+-permeable, Trpc2-dependent cation channel (19). Ca2+ entry through this channel mediates both negative and positive VSN feedback regulation through calmodulin-dependent sensory adaptation and down-regulation of the Trpc2 channel (20) and through activation of Ca2+-activated cation (20, 21) and chloride channels (22). However, the function of G proteins in this cascade has not been rigorously examined thus far. Although one study using a global deletion of Gαi2 supported this model in apical VSNs (23), no functional measurements in single VSNs were performed, and the role of Gαo in transduction of basal VSNs has not been experimentally examined. Other investigations have implicated the G protein Gαq/11 in VSN signal transduction of both subsystems (24, 25). Either molecule could contribute to sensory signaling.

We have used gene targeting to assess the role of Gαo in vomeronasal function. Gαo is widely expressed in the body, and mice harboring a global Gαo deficiency display severe neurological defects, including impaired motor control, hyperactivity, hyperalgesia, and shortened lifespan (26), thus precluding the analysis of VNO-dependent behaviors in these animals. To overcome this limitation, we created conditional Gαo null mice. These mice show a striking reduction in the sensory response of V2R-positive VSNs, indicating that Gαo is essential for signaling, subsequent membrane depolarization, and Ca2+ influx in these neurons. Behavioral analysis of the mutant mice provides insight into the logic underlying neuronal processing of molecular cues that promote aggressive interactions in both male and female mice.

Results

Using the Cre-loxP System to Create Conditional Gαo Null Mice.

To examine the role of Gαo in VSN function as well as the role of the Gαo-expressing vomeronasal subsystem in mediating instinctive behaviors, we used the Cre-loxP system to generate mice with a tissue-specific deletion of Gαo. Two Gαo splice variants have been identified: Gαo1 is encoded in exons 1–6 plus 7.1, 8.1, and 9, and Gαo2 is encoded in exons 1–6 plus 7.2 and 8.2 of Gnao1 (2628). To prevent synthesis of both forms of Gαo, two loxP sequences were inserted flanking exons 5 and 6 to generate floxed Gnao1 mice (Gnao1fx/fx) (Fig. S1). These mice were crossed with a mutant mouse line, olfactory marker protein (OMP)-Cre, in which the coding region for OMP is replaced by that of Cre recombinase (29). More breeding established offspring in which Cre-mediated excision of the target locus results in the disruption of both Gαo variants in all OMP-positive cells, including all basal VSNs. Experimental analyses were carried out in offspring homozygous for floxed Gnao1 and heterozygous for both Cre and Omp, hence-forward referred to as conditional Gαo mutants (cGαo−/−). These mice appeared healthy, survived to adulthood, and were fertile. Gnao1fx/fx (homozygous for both floxed Gnao1 and OMP) or C57BL/6J (B6) mice were used as controls.

Using RT-PCR, we identified both Gαo variants in whole VNO tissue of control mice; quantitative (q) RT-PCR analysis in this tissue showed that Gαo1 is ~30-fold more frequent than Gαo2 (Fig. S2 A and B). Using a specific antibody that recognizes both Gαo variants, we found that Gαo immunoreactivity is absent in VNO sensory epithelium and posterior (caudal) AOB of Gαo mutants, indicating that expression of Gαo is abolished in VSNs of these mice (Fig. 1A). To assess the tissue specificity of the Gαo deletion, we performed qRT-PCR analyses on VNO tissue from cGαo−/− mice and found that Gαo1 expression (copies per 1 μg total RNA) was strongly reduced compared with control tissue, whereas its expression in other tissues such as main olfactory epithelium (MOE) and whole brain was not significantly altered (Fig. 1B and Fig. S2). Gαo2 expression was relatively low in all three tissues (Fig. S2E). The absence of a significant change in Gαo transcripts in MOE of cGαo−/− mice suggests an overall low expression of Gαo in olfactory sensory neurons (OSNs) vs. VSNs (both of which express OMP) (1). RT-PCR experiments verified the presence of mRNA for several V2Rs (Vmn2r1, Vmn2r26, and Vmn2r116) and formyl peptide receptors (Fpr-rs1 and Fpr-rs7; see below) in VNO of both B6 and cGαo−/− mice (Fig. S3).

Fig. 1.
Patterns of protein expression in B6 and cGαo−/− mice. (A) Loss of Gαo immunoreactivity in VNO and AOB (encircled) of cGao−/− mice (Right). In B6 control mice, Gαo immunoreactivity (green) is present ...

Previous work using mice harboring a global Gαo deletion indicated a role for Gαo in survival of basal VSNs (30). We used immunohistochemistry in cGαo−/− mice with antibodies against V2R2 (specific for family C V2Rs that are broadly expressed in almost all basal VSNs) (31) and phosphodiesterase 4A (PDE4A; specific for the apical, Gαi2-expressing zone) (32) to show that the number of VSNs of the basal layer was approximately reduced by one-half, whereas that of the apical layer remained unchanged (Fig. 1 CE). Thus, a loss of Gαo function causes a selective loss of V2R-expressing VSNs in the basal layer of the VNO. A partial loss of VSNs has also been observed in Trpc2-deficient mice (13).

Sensory Function of V2R-Positive VSNs Requires Gαo.

The loss of V2R-positive VSNs could be secondary to a loss of sensory function caused by the Gαo deletion (30). To define the role of Gαo in VSN transduction and determine whether loss of Gαo causes alterations in ligand-evoked receptor potentials, we recorded local field potentials (EVG) from the surface of the sensory epithelium using an intact VNO preparation (32, 33). Members of at least three distinct peptide and protein families are known to be detected by V2R/Gαo-expressing VSNs: (i) peptide antigens that bind MHC proteins (17, 32), (ii) exocrine gland-secreting peptides (ESPs) (18, 34), and (iii) major urinary proteins (MUPs) (35). We, therefore, stimulated the VNO successively with prototypical members of each family—SYFPEITHI, recombinant ESP1, and recombinant MUP25—and then analyzed EVG responses in B6 vs. cGαo−/− mice. B6 controls showed robust responses to each of these stimuli (Fig. 2 A and B). In contrast, such responses were severely diminished or absent in cGαo−/− mice (Fig. 2 A and B). Importantly, this effect of the Gαo deletion was specific for V2R ligands; the magnitude of responses to other ligands such as 2-heptanone and isobutylamine, which are known to activate V1R/Gαi2-expressing VSNs (15, 16, 33), was not altered in the cGαo−/− mice, indicating that the Gαo deletion does not affect signaling in apical VSNs (Fig. 2 A and B).

Fig. 2.
Essential role of Gαo in molecular sensing by V2R-expressing VSNs. (A and B) Local field potentials (EVG) generated by VSNs of B6 and cGαo−/− mice in response to 500-ms pulses of SYFPEITHI (10−11 M), ESP1 (10−7 ...

We next used ratiometric Ca2+ imaging with Fura-2/AM in single dissociated VSNs (35) to determine whether Gαo is required for ligand-evoked Ca2+ responses in basal VSNs. These experiments essentially yielded the same results as the field potential recordings. The number of VSNs activated by each stimulus (SYFPEITHI, ESP1, and MUP25) was strikingly reduced in cGαo−/− mice (Fig. 2G). For example, in the case of MUP25, we failed to find any responding Gαo-deficient VSNs (Fig. 2G). To rule out that this diminished number in responding cells reflected the observed loss of V2R-positive VSNs in cGαo−/− mice, we first mapped Ca2+ responsiveness and then immunostained the cells with anti-V2R2 antibody (Fig. 2 C–F). Such post hoc staining revealed that virtually no V2R2-positive cells responded to chemostimulation in cGαo−/− mice (Fig. 2H). In VSNs from B6 controls, close to 100% of the cells recognizing SYFPEITHI, ESP1, and MUP25 were positive for V2R2 (Fig. 2H). Residual responses observed in the Gαo mutants did not match any cells labeled for V2R2, suggesting that basal VSNs are not involved in those responses (Fig. 2H). Importantly, Gαo-deficient VSNs showed normal Ca2+ transients to high K+ solution, indicating that general excitability and downstream Ca2+ entry remained intact in the absence of Gαo (Fig. 2E). Together, these data indicate that Gαo is required for signal transduction in V2R-expressing VSNs.

We also used the Ca2+ imaging assay to examine three additional stimuli that we later used for behavioral testing: (i) the high molecular weight fraction (>10 kDa) of urine from B6 males (gonadally intact, sexually naïve) containing the five native MUPs expressed in male B6 mice (B6-MUPs) (36, 37), (ii) the fraction of this urine comprising molecules of low molecular weight (LMW; <10 kDa, devoid of MUPs and containing small peptides and volatiles), and (iii) whole male B6 urine. We found that the vast majority of cells activated by B6-MUPs were V2R-positive; these responses depended on Gαo (Fig. 2 G and H). By contrast, LMW fraction and whole urine activated both V2R-positive and -negative VSNs; only a portion of those responses (i.e., those responses occurring in V2R-positive cells) depended on Gαo (Fig. 2 G and H).

Gαo Is Required for the Sensing of Mitochondrial N-Formylated Peptides by VSNs.

FPRs are members of a class of GPCRs that recognize formylated peptides from bacteria or mitochondria (38). Previously known for their role in the immune response, they have recently been shown to be expressed in a subpopulation of VSNs (39, 40), raising the possibility that VNO FPRs contribute to the assessment of health status during social communication. Of the five members of the FPR family that are expressed in the mouse VNO, Fpr-rs1 has been shown to be consistently coexpressed with Gαo, whereas the remaining four receptors colocalize with Gαi2 (39, 40). Gαo could be required for sensory function of VSNs expressing Fpr-rs1. To stimulate such cells, we used a specific mitochondria-derived peptide, ND1-6T (f-MFFINTLTL; derived from NADH dehydrogenase), that activates Fpr-rs1. Using EVG recordings in intact VNO, we found that f-MFFINTLTL elicits robust potentials in native B6 VSNs (Fig. 3A). We observed similar responses when we tested the related peptide ND1-6I (f-MFFINILTL) (Fig. 3A). Importantly, these peptides evoked only small residual responses in VSNs from cGαo−/− mice (Fig. 3 B and C). On average, the size of the EVG response to f-MFFINTLTL and f-MFFINILTL in cGαo−/− mice was reduced to about 10% of control mice (Fig. 3C). By contrast, the amplitude of EVG responses to f-MLF, a formyl peptide activating VNO FPRs coexpressed with Gαi2 (40), was not significantly altered in the cGαo−/− mice compared with B6 controls (P = 0.49) (Fig. 3 B and C). Live-cell Ca2+ imaging in dissociated VSNs confirmed that activation by f-MFFINTLTL in Gαo-deficient VSNs was severely reduced or absent compared with controls. Hence, we conclude that Gαo is not only required for signaling in those VSNs that express V2Rs but is also an essential requirement for the sensing of at least two N-formylated, mitochondrially encoded peptides.

Fig. 3.
Essential role of Gαo in vomeronasal recognition of mitochondrial formyl peptides. (A and B) Local field potentials generated in the VNO of B6 (A) and cGαo−/− mice (B) in response to 500-ms pulses of f-MFFINTLTL, f-MFFINILTL, ...

Gαo Mutant Males Are Less Aggressive.

To define the role of Gαo in VNO function further, we investigated innate aggressive behaviors in mice (12, 13, 23, 35, 41). We first tested male–male territorial aggression using a resident–intruder paradigm in which castrated males that no longer stimulate aggression in other males were swabbed on their backs with aggression-promoting molecular cues (35). Previous work established that both B6-MUPs as well as LMW fraction are sufficient to promote male–male aggression in this assay (35). Given that cGαo−/− mice reveal a striking reduction in the vomeronasal response to MUPs (Fig. 2) and that MUPs seem to be exclusively detected by VSNs (35), MUP-promoted male–male aggression should also be affected by the deletion of Gαo. Indeed, we found severe alterations in all measures of aggression, including attack duration, number of attacks, and latency to first attack in cGαo−/− mice vs. B6 controls (P < 0.001) (Fig. 4 A–C). The overall duration of attacks was nearly 15-fold higher for B6 vs. cGαo−/− mice.

Fig. 4.
Loss of male–male aggression in cGαo−/− mice. (A–C) Analysis of attack duration (A), number of attacks (B), and latency to first attack (C) in cGαo−/− males (n = 19) vs. B6 males (n = 22). ...

Importantly, the aggression evoked by LMW fraction was also severely diminished in cGαo−/− mice vs. control animals (P < 0.001) (Fig. 4 A–C). Quantitatively, the effects of the Gαo deletion on male–male aggression promoted by either LMW fraction or B6-MUPs were closely similar (Fig. 4 A–C). We obtained essentially the same results when we used whole urine as aggression-promoting stimulus (Fig. S4A). Together, these results indicate that Gαo is an essential requirement for the display of territorial male–male aggression in mice, independent of whether this aggression is promoted by the sensing of MUPs, LMW fraction of urine, or whole urine.

Male mice deficient in the primary VSN transduction channel Trpc2 display a high frequency of intermale mounting that persists even when males are confronted with a male–female choice of sexual partner (12, 13). This result prompted us to test whether intermale mounting is also affected by the Gαo deletion (Fig. 4D). We examined mounting behavior of male cGαo−/− mice to castrated male intruders swabbed either with male urine or PBS. Intermale mounting frequency exhibited by Gαo mutants during three 10-min test periods did not significantly differ from that of B6 controls (P = 0.85) (Fig. 4D).

Gαo is present in the axons of canonical OSNs (42), and mutant mice lacking key components of the OSN signal transduction machinery fail to show aggressive behaviors (43). Therefore, we sought to determine whether the low level of male–male aggression exhibited by the cGαo−/− mice could be a consequence of a deficit in detecting or discriminating volatile odors required for chemo-investigation. We first examined sniffing times of resident males to an intruder during the resident–intruder assay (Fig. S4B). There was no significant difference in sniffing duration between cGαo−/− males vs. B6 controls (ANOVA: F1,27 = 0.361, P = 0.55), regardless of whether castrated animals were swabbed with whole urine, LMW fraction, or B6-MUPs (ANOVA: F2,37 = 1.928, P = 0.16) (Fig. S4B). Second, we explored whether the Gαo deletion causes a deficit in a food-finding test. Although there was a slight tendency of the Gαo mutants vs. B6 controls to require more time in this test, this effect was not statistically significant (P = 0.57) (Fig. S4C). Third, we compared Gαo mutants with B6 controls in a habituation–dishabituation assay—which allows for measurement of novel odor investigation, short-term odor learning, and odor discrimination—but found no significant difference between the two genotypes (P = 0.16–0.87) (Fig. S4 D and E). Thus, the cGαo−/− mice do not display any obvious defects with respect to main olfactory system function, ruling out that the observed behavioral changes are caused by alterations in OSN function.

Defective Maternal Aggression in Gαo Mutant Females.

Aggression of lactating female mice to intruder males (maternal aggression) is strongly dependent on a functional VNO (12, 44). Previous experiments using genetically altered mice have associated maternal aggression with the function of V1R receptors (15) and Gαi2-positive VSNs (23). It came as a surprise, therefore, when we found that all measures of maternal aggression—attack duration, number of attacks, and latency to first attack—were drastically altered in cGαo−/− females vs. B6 controls, indicating that the display of maternal defense behavior is severely reduced or absent in the mutant mice (P < 0.01) (Fig. 5 A–C). We observed that both LMW fraction from male urine and B6-MUPs failed to induce aggression in cGαo−/− females, whereas B6 females exhibited aggression to both stimuli (Fig. 5 A–C). By contrast, other aspects of maternal behavior such as pup retrieval were normal in cGαo−/− females (P = 0.09–0.81) (Fig. 5D), which is in accord with studies indicating that maternal pup retrieval does not require a functional VNO (41). These results show that MUPs can initiate aggression when detected by lactating females and that this form of aggression depends on Gαo.

Fig. 5.
Loss of maternal aggression in lactating cGαo−/− females. (A–C) Analysis of attack duration (A), number of attacks (B), and latency to first attack (C) in cGαo−/− (n = 7) vs. B6 females (n = 8). ...

Discussion

These studies identify Gαo as the primary G-protein α-subunit mediating the detection of peptide and protein cues by sensory neurons of the VNO. The expression of Gαo in the VNO is restricted to the basal layer of the VNO sensory epithelium correlated with the expression of V2R receptors (810) and at least one FPR (39). By contrast, V1Rs (7), the remaining FPRs (39, 40), and some olfactory receptors (45) are expressed in the apical layer of the VNO, coexpressing Gαi2. Our results indicate that the presence of Gαo in basal VSNs is an essential and nonredundant requirement for efficient signaling in V2R-positive VSNs, leading to alterations in membrane potential and Ca2+ entry in these neurons (Fig. 2). Moreover, our data suggest that Gαo could also be essential for signal transduction in those VSNs that express the formyl peptide receptor Fpr-rs1 (Fig. 3). These cellular phenotypes are accompanied by striking alterations in behavior. Both male–male aggression (Fig. 4) and maternal aggression (Fig. 5) are severely reduced or absent in the mutant mice, whereas odor-guided behaviors depending on a functional main olfactory system (Fig. S4) seem normal in these animals. Therefore, our results reveal that Gαo is an essential requirement for the neural coding of chemosensory cues that promote aggressive interactions in both male and female mice.

Despite the presence of other G proteins in VSNs such as Gαq/11 (24, 25), single V2R2-positive VSNs from the null mice failed to exhibit significant Ca2+ signals to selected peptide and protein cues. By contrast, the Gαo-deficient VSNs showed normal Ca2+ transients in response to high K+ solution, indicating that general excitability and downstream Ca2+ signaling mechanisms remained intact in these cells. Therefore, we conclude that Gαo represents a key element in the coupling of activated V2R receptors to the downstream signal transduction machinery in VSNs. However, we cannot yet determine how this coupling exactly occurs in intact VSNs. Although there is good evidence that PLC represents the downstream key enzyme in this cascade (14, 19, 46), conclusive proof that the PLCβ2 isoform mediates VSN signaling (46) will require a gene deletion approach similar to the approach developed here. In any case, Gαo is unlikely to couple with PLCβ2, because no such interaction has ever been identified. Hence, it has been suggested that Gβγ, which does activate PLCβ2 (47), mediates this molecular step (48).

Gαo is the most abundant G protein in the nervous system, and mice with a global Gαo deficiency exhibit a wide variety of defects that span from motor control to pain perception (26). These mice, therefore, do not provide an adequate animal model for the investigation of VNO-dependent behavioral changes. To circumvent this problem, we created a conditional KO model in which the Gαo deletion was restricted to OMP-positive cells. Importantly, the conditional null mice showed no obvious defects in odor-guided behaviors that depend on a functional main olfactory system in contrast with studies using mice with a global Gαo deficiency (49), indicating that the observed behavioral deficits are caused by the described changes in VNO sensing. We then focused specifically on the analysis of aggressive behaviors, because these behaviors have been clearly established to depend on intact VNO function (12, 13, 15, 23, 35, 41). We drew three main conclusions regarding the relative roles of Gαo- and Gαi2-expressing VNO subsystems in the coding of molecular cues that promote aggressive interactions.

First, chemostimuli influencing the aggressive state of mice are detected by widely distributed VSN populations that are localized in both apical and basal layers of the VNO. With respect to maternal aggression, deletion of a cluster of V1R receptors substantially altered the level of aggression in lactating females (15). Consistent with this finding, the display of maternal aggression was also severely blunted in Gαi2 mutant mice (23). Surprisingly, our present findings showed that maternal aggression was similarly affected after deletion of Gαo, and an analogous situation exists for male–male territorial aggression, where the deletion of either Gαi2 (23) or Gαo (Fig. 4) severely altered the display of intermale aggression.

Second, given that VSNs in both layers detect distinct types of molecular cues (1), diverse chemicals must be involved in aggression-promoting activity, those chemicals that act on V1Rs in Gαi2-positive VSNs and those chemicals that are detected by V2Rs in Gαο-positive VSNs. Examples for each case have indeed been found. When added to castrated males, MUPs, which activate V2R-positive VSNs, provoked aggressive behavior [ref. 35 and this study]. Similarly, when spiked in castrated urine, two volatile urine constituents, dehydro-exo-brevicomin and 2-sec-butyl-dihydrothiazole (which are both detected by apical VSNs) (33), provoked intermale fighting (50).

Third, although activation of apical or basal VSNs by distinct cues was sufficient to induce aggression, the two populations of VSNs somehow seem to work in concert to promote aggression. This finding is indicated by a loss of aggression after loss of function of either apical or basal VSN subsystem [ref. 23 and this study] and by our finding that stimulation with LMW fraction or even whole urine did not promote aggression in the Gαo-deficient mice. Therefore, our results support a model (Fig. 5E) that is based on multiple aggression-promoting circuits—each circuit required for the full display of behavioral outcomes—and in which information sensed by apical and basal VSN populations is eventually integrated to generate an aggressive response. The conditional Gαo null mice may ultimately contribute to the precise identification of functional neuronal circuits (51, 52) underlying the control of mammalian instinctive behaviors such as aggression.

Materials and Methods

Mice.

Animal care and experimental procedures were performed in accordance with the guidelines established by the animal welfare committee of the relevant institution. Details on generation of conditional null mice, immunohistochemistry, stimulus delivery, electrophysiology, Ca2+ imaging, and behavioral testing are in SI Materials and Methods.

RT-PCR Analyses.

VNO, MOE, and whole brain were dissected from cGαo−/− and B6 mice, and RNA in each tissue was isolated with the InnuPREP RNA isolation kit (Analytik Jena) and assessed by gel electrophoresis and photometric measurements. PCR products were amplified with gene-specific primers, and specificity was controlled by sequencing. Additional details are in SI Materials and Methods.

Statistical Tests.

Data were analyzed using NCSS 2004 statistical software. The Mann–Whitney u test, a nonparametric inferential statistic, was used to determine whether two groups differ significantly. The unpaired student t test was used for measuring the significance of difference between two independent distributions. Multiple groups were compared using a two-way ANOVA with the Fisher's least significant difference (LSD) as a post hoc comparison. An ANOVA for multiple repeated measurements was used in experiments using repeated measurements (Figs. 4 and and55 and Fig. S4). Unless otherwise stated, results are presented as means ± SEM.

Supplementary Material

Supporting Information:

Acknowledgments

We thank P. Mombaerts for supplying OMP-Cre mice, K. Touhara for the ESP1 plasmid, R. Tirindelli for the V2R2 antibody, and M. Pyrski and T. Meier for help with genotyping. This work was supported by Deutsche Forschungsgemeinschaft Grants CH 920/2-1 (to P.C.), SFB 530 (to F.Z.), SFB 894 (to F.Z.), and SFB 894 (to T.L.-Z.), Intramural Research Program of the National Institutes of Health Project Z01 ES-101643 (to L.B.), National Institutes of Health Grant DC010857 (to H.M.), and the Volkswagen Foundation (to T.L.-Z.). Also, T.L.-Z. is a Lichtenberg Professor of the Volkswagen Foundation.

Footnotes

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1107770108/-/DCSupplemental.

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