Essential Role of the Main Olfactory System in Social Recognition of Major Histocompatibility Complex Peptide Ligands

Nonvolatile molecules gain access to the MOE

Social investigation in mice involves periods of robust physical contact between conspecifics, during which intense sniffing and licking of facial and anogenital areas occurs. We hypothesized that nonvolatile chemostimuli may gain access to the MOE during such behaviors. To test this, we used a dye-tracing approach (Wysocki et al., 1980; Leinders-Zufall et al., 2004). Female urine was supplemented with the nonvolatile fluorescent dye rhodamine, and a drop was placed on a male’s ONG. Alternatively, the urine–rhodamine mixture was placed in the AGR of an estrous female, and males were allowed to freely investigate. After sniffing or anogenital investigation, we examined rhodamine fluorescence in the nasal cavity. To exclude false-positive results attributable to preparational artifacts (i.e., crushing or squeezing the dye-containing VNO), we used mice that had their VNOs surgically removed (VNX) (Wysocki and Wysocki, 1995; Pankevich et al., 2004). Both placement on the ONG (Fig. 1A,B) and application to a female’s AGR (Fig. 1C,D) caused strong rhodamine fluorescence throughout the endoturbinate system of the MOE. The same results were observed in mice that had not undergone surgery. However, such fluorescence was never observed when normal rhodamine-free urine was used (Fig. 1E,F). These results reveal that nonvolatile compounds present in urine and other bodily secretions gain access to the MOE during behavioral situations involving direct physical contact.

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Nonvolatile molecules gain access to the mouse MOE after direct contact with the stimulus source. A–F, Bright-field (A, C, E) and fluorescence images (B, D, F) showing the dissected endoturbinate system of male C57BL/6 mice. Female urine was placed on a male’s oronasal groove (ONG) (A, B, E, F) or in the anogenital region (AGR) of an estrous female, and males were allowed to freely investigate (C, D). When the urine contained the nonvolatile rhodamine dye (0.01%), rhodamine fluorescence was visible on all four endoturbinates (B, D), whereas control animals treated with rhodamine-free urine did not show such fluorescence (F). Fluorescence images in B, D, and F were taken under identical optical conditions. No significant autofluorescence was observed at this wavelength. All of these experiments used VNX mice. Roman numerals designate individual turbinates. a, Anterior; p, posterior; d, dorsal; v, ventral. Scale bars, 500 μm.

MHC class I peptides are potent stimuli for sensory neurons in the MOE

Murine MHC class I peptide ligands are nonvolatile molecules, typically nine amino acid residues long, that are presented by MHC molecules at the cell surface. When peptide/MHC complexes are released into the extracellular space and appear in urine and other bodily secretions (Singh et al., 1987), any information contained in their chemical complexity becomes a property of the entire individual (Boehm et al., 2003; Boehm and Zufall, 2005). Given the results of our dye-tracing studies (Fig. 1), these peptides could potentially function as social recognition signals detected by the MOE. Are MHC peptides thus capable of stimulating sensory neurons in mouse MOE? To examine this question, we recorded local field potentials (the EOG) from the ciliated surface of the MOE using methods that do not require olfactory stimuli to be volatile (Chen et al., 2000; Munger et al., 2001) and tested whether known peptide ligands of MHC class I molecules elicit an electrical response in olfactory sensory neurons (OSNs). We chose peptides that prototypically correspond to two disparate H-2 haplotypes (Leinders-Zufall et al., 2004), namely AAPDNRETF (for the H-2D^b haplotype of C57BL/6 mice) and SYFPEITHI (for the unrelated H-2K^d haplotype of BALB/c mice) (Fig. 2A). Both representative peptides evoked negative field potentials in a dose-dependent manner in the MOE of male and female C57BL/6 mice carrying the H-2D^b haplotype (Fig. 2B), suggesting that cognate MHC class I molecules are not required for this response. For both peptides, we found detection thresholds at concentrations near or below 10⁻¹¹ m (Fig. 2B,C). These results reveal an exquisite sensitivity of the mouse MOE for MHC class I peptides.

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The mouse MOE detects MHC class I peptide ligands. A, Peptide ligands for prototypical representatives of MHC class I molecules used in this study. Sequences (in one-letter code) and sources were taken from Rammensee et al. (1997), which provides detailed primary references. Anchor residues defining haplotype-specific binding characteristics are highlighted in bold, and mutated residues in control peptides are underlined. In the ANPRAFDTE peptide, anchor positions 5 and 9 of the WT peptide were replaced with residues unfavorable for binding to the D^b MHC molecule (Rammensee et al., 1997), whereas the overall amino acid composition remains identical. minor H Ag, Minor histocompatibility antigen; TK, tyrosine kinase. B, Examples of peptide-evoked MOE field potentials and their dose dependence (each series from a single location) to varying concentrations of either AAPDNRETF (representative of 43 recordings in 5 animals) or SYFPEITHI (representative of 38 recordings in 12 animals). Responses are produced by 500 ms pulses of peptides as indicated. C, Average dose–response plots of peak responses to AAPDNRETF (filled circles, black curve) and SYFPEITHI (open circles, red curve). The results for two control peptides, AAPDARETA (solid triangles) and SAFPEITHA (open triangles), respectively, are also shown. Smooth curves are fitted by the Hill equation, with K_1/2 values and Hill coefficients of 190 pm, 0.6 (SYFPEITHI); 240 pm, 0.6 (AAPDNRETF); 5.4 nm, 0.8 (SAFPEITHA); and 8.3 nm, 1.3 (AAPDARETA). D, Medial view of the mouse endoturbinate system after dissection. Roman numerals designate individual turbinates. Sixty spatial locations of 96 spots stimulated that exhibited a field potential response to AAPDNRETF and/or SYFPEITHI at 10⁻¹⁰ m are shown. The other 36 locations that did not respond are included in the map in Figure 3A, which documents the entire data set. Scale bar, 1000 μm. E, F, Specificity of peptide-evoked MOE field potentials assessed by two control peptides in which the characteristic anchor residues of MHC class I ligands were replaced by alanines [i.e., AAPDARETA (8 recordings, 3 animals) and SAFPEITHA (14 recordings, 4 animals), respectively]. These peptides failed to elicit a field potential at 10⁻¹⁰ m (E). A scrambled version of the H2-D^b ligand AAPDNRETF, ANPRAFDTE (10⁻¹⁰ m), and a mixture containing all amino acids (aa mix; in free form, each at 10⁻¹⁰ m) that constitute the SYFPEITHI peptide elicited only very small or no responses (E, F). Each pair of responses in E corresponded to the same spatial location. Error bars indicate SEM.

For comparison, we tested the effect of the urinary volatile 2-heptanone, one of the prospective pheromones that act as sensory cues in both the MOE and VNO (Leinders-Zufall et al., 2000; Boschat et al., 2002; Ziesmann et al., 2002; Xu et al., 2005), and found that this stimulus also elicits MOE field potentials at 10⁻¹⁰ m (Figs. 3B, 4C). Likewise, other urinary volatile chemosignals such as 2,5-dimethylpyrazine, 2-sec-butyl-4,5-dihydrothiazole, 2,3-dehydro-exo-brevicomin, and 6-hydroxy-6-methyl-3-heptanone are capable of evoking sensory responses at subnanomolar concentrations in the MOE (Ziesmann et al., 2002) as well as the VNO (Leinders-Zufall et al., 2000). Boschat et al., 2002; Ziesmann et al., 2002; Xu et al., 2005), and found that this stimulus also elicits MOE field potentials at 10⁻¹⁰ m (Figs. 3B, 4C). Likewise, other urinary volatile chemosignals such as 2,5-dimethylpyrazine, 2-sec-butyl-4,5-dihydrothiazole, 2,3-dehydro-exo-brevicomin, and 6-hydroxy-6-methyl-3-heptanone are capable of evoking sensory responses at subnanomolar concentrations in the MOE (Ziesmann et al., 2002) as well as the VNO (Leinders-Zufall et al., 2000).

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Spatial distribution of peptide-evoked field potentials on endoturbinates IIa, IIb, III, and IV. A, Map of 96 spatial locations that were stimulated sequentially with AAPDNRETF, SYFPEITHI, and 2-heptanone (each at 10⁻¹⁰ m). Color-coded symbols indicate whether a given location responded to none, one, two, or all three of these stimuli. Scale bar, 1000 μm. B, Examples of field potential responses registered in different locations. The numerals (1–7) refer to a given location as shown in A. Ejecting bath solution (control) onto the MOE gave no responses. C, Analysis of the data shown in A with respect to the frequency of observing a response to each of the three ligands (at 10⁻¹⁰ m) on a given endoturbinate (n, number of locations tested on each turbinate).

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Recognition of MHC peptides by the MOE requires cAMP signaling and the CNG channel subunits A2 and A4. A, Inhibition of a peptide-induced field potential by SQ22536 (300 μm). B, Summary of the effects of pharmacological blockers on peptide-evoked potentials. Each response was normalized to its own control response obtained before application of each drug as shown in A. C, Representative peptide-induced responses from the MOE of WT and CNGA2^−/− mice. Responses to 2-heptanone (10⁻¹⁰ m) are shown for comparison. In accord with Lin et al. (2004), we observed residual EOG responses to 2-heptanone in CNGA2^−/− mice at a much higher concentration (10⁻⁴ m; n = 9) (data not shown). D, Average size of peptide-evoked MOE potentials in mice deficient for CNGA2 or CNGA4 compared with WT littermate controls. E, Representative responses to a prolonged 6 s peptide pulse in WT and CNGA4^−/− MOE. Fit is a single-exponential decay with a time constant (τ) of 1.1 s. F, G, Diminished adaptation rate of peptide-evoked potentials in CNGA4^−/− MOE as observed by measuring plateau/peak ratios (F) or desensitization time constants (G), respectively (means ± SEM; Student’s t test; **p < 0.001). The peptide-evoked potentials were recorded on endoturbinate III in response to SYFPEITHI (10⁻¹⁰ m). The number of experiments is indicated above each bar. See Results for rationale.

Figures 2D and 3 examine the spatial distribution of peptide responses in the MOE in more detail. We sequentially stimulated 96 different locations on all four endoturbinates with AAPDNRETF and SYFPEITHI as well as 2-heptanone (each at 10⁻¹⁰ m). By superimposing the locations of peptide-evoked potentials onto a bright-light image of the MOE, we produced coarse maps of the spatial organization of peptide sensitivity (Figs. 2D, 3A). Of these 96 locations, 54% (52 of 96) responded to the D^b ligand, and 60% (58 of 96) responded to the K^d ligand (Fig. 3A–C). 2-Heptanone evoked field potentials in 37% (35 of 96) of these locations (Fig. 3A–C). Collectively, these results demonstrate that sensitive peptide recognition is widely distributed in the MOE and occurs on all four endoturbinates. The fact that some locations showed a high sensitivity whereas others did not respond to these stimuli (Fig. 3B) is broadly consistent with the mosaic organization of the mouse MOE as reported by Ma and Shepherd (2000).

To assess the specificity of peptide detection by the MOE, we used two mutated control peptides. In these ligands, the characteristic anchor residues, which fit into specific binding pockets of the MHC molecule and are thus a defining feature of an MHC peptide (Rammensee et al., 1997), were replaced by alanines (i.e., AAPDARETA and SAFPEITHA, respectively). These peptides failed to elicit MOE responses at 10⁻¹⁰ m (Fig. 2E) and showed dose–response curves that are shifted to higher concentrations by approximately two logarithmic units (Fig. 2C). Furthermore, a scrambled version of the H-2D^b ligand AAPDNRETF, ANPRAFDTE (10⁻¹⁰ m), and a mixture containing all amino acids (in free form, each at 10⁻¹⁰ m) that constitute the SYFPEITHI peptide elicited only very small or no responses (Fig. 2E,F). Thus, peptides must meet precise structural specifications to induce low-threshold activation of sensory neurons in the MOE.

Peptide detection by the MOE requires canonical cAMP signaling

To begin to identify the cellular and molecular mechanisms underlying peptide detection by the MOE, we next asked whether the canonical cAMP second-messenger system found in the main population of OSNs (Firestein, 2001; Zufall and Munger, 2001) is required for peptide-evoked field potentials. Using SYFPEITHI (10⁻¹⁰ m) as a stimulus, we found that responses are inhibited in a reversible manner by application of an antagonist of adenylyl cyclase, SQ22536 (Chen et al., 2000) (300 μm) (Fig. 4A,B). Another adenylyl cyclase antagonist, MDL12330A (Chen et al., 2000) (50 μm), produced very similar results indicating that cAMP formation is required for these responses (Fig. 4B). In contrast, 2-APB (50 μm), which blocks Ca²⁺-permeable, diacylglycerol-activated cation channels essential for VNO transduction and inhibits peptide-evoked potentials in the VNO (Lucas et al., 2003; Leinders-Zufall et al., 2004), did not significantly reduce the size of peptide-induced MOE responses (Fig. 4B). Hence, MHC class I peptides are detected in the MOE by sensory neurons using transduction mechanisms that are distinct from those in the VNO.

To further support this notion, we next recorded MOE field potentials from mice exhibiting targeted deletions in specific genes essential for cAMP-dependent sensory transduction in OSNs. Results were compared with those from WT littermates. Mice deficient for CNGA2 (Zhao and Reed, 2001), which encodes the principal subunit of the olfactory cyclic nucleotide-gated (CNG) cation channel, failed to respond to SYFPEITHI (10⁻¹⁰ m; n = 26) (Fig. 4C,D). The same result was obtained when we used AAPDNRETF (10⁻¹⁰ m; n = 12) (data not shown) or 2-heptanone (10⁻¹⁰ m; n = 12) (Fig. 4C) as stimuli. If peptide-evoked EOG responses are mediated by signal transduction events in the main population of OSNs, these responses should not only depend on CNGA2 but also be impaired in CNGA4^−/− mice, which lack a modulatory subunit of the olfactory CNG channel and exhibit specific defects in odor adaptation (Munger et al., 2001; Kelliher et al., 2003). Indeed, peptide-evoked potentials from CNGA4^−/− MOE displayed a characteristic reduction in the desensitization rate compared with those from WT mice (Fig. 4E–G). Hence, we conclude that MHC class I peptides, in the MOE, are transduced by OSNs using a cAMP-signaling pathway and the canonical olfactory CNG channel.

Single peptide-sensitive OSNs in MOE slices

Next, we investigated peptide recognition in the MOE at the single-cell level by using an in situ Ca²⁺ mapping approach (Ziesmann et al., 2002). Acute coronal MOE slices (C57BL/6 mice) were loaded with the Ca²⁺ indicator fluo-4/AM and imaged at an optical resolution sufficient to detect responses in single OSNs (Fig. 5). Stimulation with AAPDNRETF or SYFPEITHI (each at 10⁻⁹ m) produced transient increases in intracellular Ca²⁺ in specific subsets of OSNs when tested in such slices (Fig. 5A–D). Peptide-sensitive cells were widely distributed in the MOE and found in the epithelium covering the dorsal recess (Fig. 5A,C), endoturbinates I and IIa, and ectoturbinates 1 and 2. In total, we imaged 890 OSNs (13 slices, 8 mice), of which 26 cells (2.9%) responded to either of these two peptides. In contrast to previous findings in the VNO (Leinders-Zufall et al., 2004), we did not observe cells that responded to both of these two peptides. Of these 890 OSNs, 16 (1.8%) responded to the D^b ligand and 10 responded (1.1%) to the K^d ligand. After peptide stimulation, we applied the adenylyl cyclase activator forskolin (20 μm) to demonstrate that peptide-sensitive OSNs exhibit a cAMP-dependent Ca²⁺ rise (Fig. 5D) (n = 12), consistent with the results described in Figure 4.

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In situ Ca²⁺ mapping reveals distinct populations of peptide-sensitive OSNs. A, Transmitted light image of a mouse coronal MOE slice (P7). DR, Dorsal recess; S, nasal septum. A portion of the sensory epithelium delimited by the white box is shown at a higher magnification in B and C. B, Confocal fluorescence image of the fluo-4-loaded MOE acquired at rest (grayscale). C, The same region is depicted as a merged pseudocolor image of the relative increase in peptide-induced Ca²⁺-dependent dye fluorescence (ratio between the peak fluorescence before and after stimulation, ΔF/F). In this example, AAPDNRETF (10⁻⁹ m; green) activated three OSNs (cells 1, 2, and 4), and SYFPEITHI (10⁻⁹ m; red) activated one OSN (cell 3). In some cases, individual somata, dendrites, and dendritic knobs can be clearly distinguished. D, Time course of peptide-induced Ca²⁺ responses from the same cells that are shown in C. The tissue was successively stimulated with five different peptides (each at 10⁻⁹ m), followed by forskolin (20 μm). Note that cells 1 and 2 each recognized two peptides with different anchor residues. The arrows indicate the time points at which stimulus application was turned on. Scale bars: A, 200 μm; B, C, 10 μm.

To further investigate sequence-specific tuning of individual OSNs, we used the scrambled peptide ANPRAFDTE and the two mutated peptides AAPDARETA and SAFPEITHA (each at 10⁻⁹ m) in a portion of our imaging experiments (n = 246 cells) (Fig. 5D). Although we did not observe any response to ANPRAFDTE or SAFPEITHA, two-thirds (six of nine) of the AAPDNRETF-sensitive cells produced a small but significant Ca²⁺ transient to AAPDARETA (Fig. 5D, cells 1 and 2). This result is not only consistent with the field potential recordings in Figure 2C but also demonstrates that at least some OSNs are capable of recognizing multiple peptides exhibiting different anchor residues. Hence, peptide recognition in the MOE likely occurs in a combinatorial manner.

MOE-dependent peptide detection in behaving mice

To determine the biological implications of our findings, we investigated whether behaving mice are capable of recognizing MHC peptides through their MOE and whether they might assign meaning to this information in appropriate social behaviors. Because the main olfactory system plays an important role in attracting males to females (Keverne, 2004), we tested social preference by giving sexually naive male mice a choice between pairs of identical female same-strain urine that were supplemented with different haplotype-specific MHC class I peptide mixtures. Stimuli were pipetted on either of two cotton-tipped applicators that were placed in a randomized manner in adjacent corners of the cage (cotton-tip test; see Materials and Methods). All mice showed a keen interest in investigating the stimulus source with intense periods of sniffing, during which their noses were in direct physical contact with the cotton tips (see supplemental movie 1, available at www.jneurosci.org as supplemental material). Preferences were calculated as a percentage of time (mean ± SEM) spent investigating the cotton tips during a 5 min test period. The histogram in Figure 6A shows that the average investigation time is relatively maintained throughout this test.

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Essential role of the main olfactory system and the CNGA2 channel subunit in the display of a social preference for disparate MHC class I peptides in male mice. A, Relative investigation time (means ± SEM) in the cotton-tip test plotted as a function of time. Bin width, 1 min. Data are based on results from animals 15–26 (preoperative) and 41–52 (see supplemental Fig. 7, available at www.jneurosci.org as supplemental material, for individual mice). For each individual animal, the investigation time per 1 min bin was measured and normalized to the maximum value. B, Preference of C57BL/6 males for urine obtained from BALB/c (gray) versus C57BL/6 (black) female mice [F_(1,27) = 124.1; Student–Newman–Keuls (SNK) test; *p < 0.001]. C, Preference of C57BL/6 males for female same-strain urine supplemented with mixtures of either BALB/c (gray) or C57BL/6 (black) peptides. A preference for disparate peptides was observed in preoperative (pre-op) and VNX mice (F_(1,47) = 29.5; SNK; *p < 0.01) with no significant effect of surgical VNX (SNK; p = 0.43). D, When direct physical contact is precluded (caged stimuli), C57BL/6 males do not show a preference for female same-strain urine supplemented with mixtures of BALB/c (gray) versus C57BL/6 (black) peptides (F_(1,27) = 2.3; SNK; ^NSp = 0.16). E, Preference of BALB/c males for female same-strain urine supplemented with C57BL/6 (black) versus BALB/c (gray) peptides (F_(1,23) = 10.0; SNK; *p < 0.01). F, Representative images of coronal sections through the main and accessory olfactory bulb of sham-operated and VNX mice. Sections were stained with HRP-SBA. The absence of HRP-SBA labeling in the accessory olfactory bulb of VNX animals signifies the complete removal of the VNO. GL/AOB, Glomerular layer of the accessory olfactory bulb. G, Preference of CNGA2^+/0 and CNGA2^−/0 males for female same-strain urine supplemented with BALB/c peptides (gray) or solvent (black). CNGA2^+/0 but not CNGA2^−/0 mice displayed a significant preference for disparate peptides (F_(1,47) = 11.1; SNK; *p < 0.01; ^NSp = 0.33).

To check for the validity of the assay, we first tested whether C57BL/6 males show a preference when presented with unaltered urine from either C57BL/6 or BALB/c females. We found that C57BL/6 males display a strong preference in investigating BALB/c versus C57BL/6 urine (Fig. 6B) (74.8 ± 2.2 vs 25.2 ± 2.2%; n = 14; p < 0.001).

Next, we tested whether C57BL/6 males (preoperative) display a preference in investigating C57BL/6 urine supplemented with peptides specific for BALB/c as opposed to C57BL/6 haplotypes. This was indeed the case (Fig. 6C, pre-op) (67.8 ± 3.9 vs 32.2 ± 3.9%; n = 12; p < 0.01), indicating that the attractiveness of same-strain urine can be enhanced by the addition of disparate MHC peptides. Importantly, this preference did not require a functional VNO, because it was still evident in the same C57BL/6 animals after surgical removal of the VNO (Fig. 6C, VNX) (62.0 ± 5.0 vs 38.0 ± 5.0%; n = 12; p < 0.01). Complete removal of the VNO was proven in histological sections of the main and accessory olfactory bulb by specific labeling of intact vomeronasal nerve fibers with HRP-SBA (Wysocki and Wysocki, 1995; Pankevich et al., 2004). Sham-operated control mice (n = 3) showed heavy HRP-SBA staining at the AOB level, whereas no staining was observed in VNX animals (n = 12) (Fig. 6F). A potential caveat of the results in Figure 6C is that the tests using VNX mice were performed with animals that had undergone previous testing. Naive animals might be more VNO dependent. However, the data shown in Figure 6G argue against this.

To control for potential changes in the release of urine volatiles after addition of the peptides to urine, we used the same paradigm as in Figure 6C (“pre-op”) under conditions that precluded direct physical access to the cotton tips without posing a barrier for the volatiles (“caged stimuli”; see Materials and Methods). No preference was observed in this case (Fig. 6D) (45.2 ± 3.2 vs 54.8 ± 3.2%; n = 14; p = 0.16), ruling out that the presence of MHC peptides somehow affects release of urine volatiles.

To demonstrate that H-2K^d peptides are not attractive per se but that this function depends on a combination with an animal’s own genotype, we performed the preference test in Figure 6C (pre-op) with BALB/c males (Fig. 6E). These animals showed a clear preference for same-strain urine supplemented with peptides specific for C57BL/6 as opposed to BALB/c haplotypes (64.1 ± 4.9 vs 35.9 ± 4.9%; n = 12; p < 0.01). Together, these results indicate that male mice display a social preference for disparate MHC peptides and that this preference does not require a functional VNO.

To further determine whether this effect depends on peptide recognition by the main population of OSNs in the MOE, we used mice deficient for the CNGA2 gene. Here, we observed a significant preference for disparate peptides in CNGA2^+/0 (70.5 ± 5.3 vs 29.5 ± 5.3%; n = 12; p < 0.01) but not in CNGA2^−/0 (54.2 ± 5.9 vs 45.8 ± 5.9%; n = 12; p = 0.33) mice (Fig. 6G).

All of these averaged behavioral results were fully supported by examining data from each individual mouse (supplemental Fig. 7, available at www.jneurosci.org as supplemental material). In these tests, we have added three exogenous peptide ligands to urine, each at a final concentration of 12.5 μm (see Materials and Methods). Importantly, our results also show that, under the conditions of the preference paradigm used here, mice are still capable of displaying a preference when the concentration of the peptide mixture was decreased by 10-fold (supplemental Fig. 8, available at www.jneurosci.org as supplemental material). Thus, on the basis of these findings, we conclude that detection of MHC peptides by OSNs in the MOE is biologically relevant for the display of a social preference in male mice and that this effect depends critically on an intact CNGA2 gene.

Access of nonvolatile cues to the MOE

A prerequisite for peptide recognition by the MOE is that these cues reach the olfactory mucosa in natural behavioral situations. A widely held view is that odorants must be sufficiently volatile to be detected by the MOE. We have used a nonvolatile fluorescent marker to demonstrate that nonvolatile molecules in urine gain access to the MOE of freely behaving mice during social investigatory behaviors that involve direct physical contact. Our results are supported by previous reports that showed transport of rhodamine-supplemented female urine halfway up the nasal septum of male guinea pigs (Wysocki et al., 1980) and uptake of HRP (40 kDa) to all areas of the MOE in male hamsters (Meredith, 1983; Meredith and O’Connell, 1988). We therefore conclude that, after direct physical contact with the stimulus source, nonvolatile constituents present in urine and other bodily secretions not only gain access to the VNO but also to the MOE.

High sensitivity of the mouse MOE

A finding of importance is that subsets of OSNs detect MHC peptides at surprisingly low concentrations. Field potential dose–response curves show EC₅₀ values near 10⁻¹⁰ m, and single OSNs respond to nanomolar doses of peptides with a Ca²⁺ rise. For comparison, we used a volatile constituent of mouse urine, 2-heptanone, and showed that this compound also evokes MOE field potentials at 10⁻¹⁰ m. Although for many odors the dose–response curves in single OSNs have relatively elevated EC₅₀ values, in the range 10–100 μm (summarized by Firestein, 2001), our results are supported by a growing body of evidence indicating that the mammalian MOE may detect some odor ligands at much lower concentrations than previously thought (Shepherd, 2004). For example, in accord with our findings, the olfactory receptor OR912-93 recognizes 10 nm 2-heptanone when expressed in a heterologous cell system (Gaillard et al., 2002). Likewise, in vivo imaging of glomerular activity in the mouse main olfactory bulb has revealed detection of short aliphatic odor molecules in the low nanomolar range (Bozza et al., 2004). It is not yet clear why the MOE exhibits high-sensitivity responses in some studies but not in others. These discrepancies could reflect differences in the recording methods (i.e., dissociated cells vs more intact preparations) or they could reflect the fact that it is often difficult to identify a preferred ligand for a given OSN.

Identity of peptide-sensitive OSNs

Although our imaging results reveal for the first time the existence of individual peptide-sensitive OSNs in the mammalian MOE, we cannot yet determine the molecular identity of these cells. Ciliated neurons of the MOE comprise a heterogeneous population, with subgroups of cells displaying unique patterns of gene expression (Zufall and Munger, 2001). One such group expresses a receptor guanylyl cyclase, GC-D (Fülle et al., 1995; Meyer et al., 2000). These cells do not express CNGA2 but rather the cGMP-specific CNGA3 channel, ruling out an involvement in the responses shown here. Another intriguing OSN subpopulation, defined by the expression of members of the OR37 subfamily, occupies a characteristic location on endoturbinate IIb (Strotmann et al., 2000). Our mapping results (Fig. 3) argue against a predominant role for OR37-expressing OSNs in MHC peptide responses. Collectively, our results show that peptide-recognizing OSNs differ in several important aspects from peptide-detecting VSNs (Leinders-Zufall et al., 2004), including the sensitivity, the signal transduction pathway, and the specificity. Detection thresholds in VSNs are approximately two orders of magnitude lower compared with those of OSNs, and peptide recognition in OSNs, unlike VSNs, involves the canonical cAMP second-messenger pathway. There are also clear differences in the specificity to MHC peptides in both types of cells. Whereas VSNs do not respond to elevated concentrations of MHC peptides in which the characteristic anchor residues are mutated (Leinders-Zufall et al., 2004), such peptides can be recognized by OSNs (Figs. 2, 5). Although our imaging results suggest a combinatorial scheme for the detection of MHC peptides by OSNs, we do not yet know the exact rules of this mechanism. Deciphering the precise tuning properties of peptide-sensitive OSNs and VSNs and identifying the receptors involved in peptide recognition remains a major goal of future research. On the basis of the considerations presented here, it seems unlikely that the same receptors are used for peptide recognition in both the MOE and VNO. It should be interesting to determine whether these receptors belong to family A (such as ORs) or family C (such as V2Rs) of G-protein-coupled receptors (GPCRs), or even other yet unknown receptor families. In this respect, it is important to note that, among known family A GPCRs, peptide receptors form a large subgroup and mediate important physiological roles of endogenous signaling peptides (Kristiansen, 2004).

Peptide recognition and the dual olfactory hypothesis

In the traditional view, known as the dual olfactory hypothesis (Scalia and Winans, 1975), the main and accessory olfactory systems form separate and parallel pathways presumably involved in the detection of distinct sets of olfactory cues (summarized by Halpern, 1987). For example, the accessory system has been widely thought to detect nonvolatile pheromones that trigger behavioral and endocrine responses, whereas the main system has generally been regarded as a nonselective molecular analyzer of volatile odorants. In contrast, a growing body of evidence suggesting far greater integration and complexity of chemosensory detection of both systems has challenged this classical view (Meredith, 1998; Xu et al., 2005, and references therein). Our results support and extend this notion by demonstrating that olfactory recognition of MHC peptides in mice occurs through both the main and accessory olfactory system. This result, however, does not mean that the functions of both systems are redundant. In fact, our behavioral data provide direct evidence for distinct functions of the two olfactory pathways in different behavioral contexts. For instance, detection for disparate MHC peptides in the social preference test depends on CNGA2 function in the MOE but is independent of a functional VNO (Fig. 6). In contrast, MOE-dependent peptide recognition in the absence of a functional VNO was not sufficient to influence the outcome of the pregnancy-block paradigm (Kelliher et al., 2005). Therefore, it appears that the same social cues may mediate different conspecific behaviors through the activation of each system. We note that our results do not exclude the role of other, particularly volatile chemosignals in behavioral decisions in mice (Restrepo et al., 2004). Indeed, a male mouse urinary volatile that can be detected by the main olfactory system has recently been shown to enhance urine attractiveness to females (Lin et al., 2005). The exact concentration of MHC peptides in urine is currently unknown. Urine might contain hundreds of peptide sequences that might be constitutive or inducible (i.e., vary over time depending on hormonal cycling, maturation stage, and other factors) (Boehm and Zufall, 2005). It is also unclear whether urine is the only source of MHC peptide ligands or whether peptides from other bodily secretions (Kimoto et al., 2005) contribute to social preference in mice. Thus, care must be taken when comparing the results from the preference test to what may occur in a real biological setting. It seems likely that social recognition under natural circumstances is vastly more complex than our controlled testing paradigm and probably involves the combined detection and integration of a variety of classes of chemosignals (Boehm and Zufall, 2005).

Outlook

Our results are of direct relevance to previous studies (Wedekind et al., 1995; Jacob et al., 2002) on the role of MHC-related odors in human behavioral decisions. Our finding that MHC peptide ligands can gain access to and be recognized by sensory neurons of the MOE will prompt further investigations aimed at understanding whether evolutionary conserved MHC peptide ligands also function as odor signals in humans.