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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2010 December 2.
Published in final edited form as:
PMCID: PMC2919682
NIHMSID: NIHMS210608

Distinct Signals Conveyed by Pheromone Concentrations to the Mouse Vomeronasal Organ

Abstract

In mammalian species, detection of pheromone cues by the vomeronasal organ (VNO) at different concentrations can elicit distinct behavioral responses and endocrine changes. It is not well understood how concentration-dependent activation of the VNO impacts innate behaviors. In this study, we find that when mice investigate the urogenital areas of a conspecific animal, the urinary pheromones can reach the VNO at a concentration of ~1% of that in urine. At this level, urinary pheromones elicit responses from a subset of cells that are tuned to sex-specific cues and provide unambiguous identification of the sex and strain of animals. In contrast, low concentrations of urine do not activate these cells. Strikingly, we find a population of neurons that is only activated by low concentrations of urine. The properties of these neurons are not found in neurons responding to putative single pheromones. Further analyses show that these neurons are masked by high concentration pheromones. Thus, an antagonistic interaction in natural pheromones results in the activation of distinct populations of cells at different concentrations. The differential activation is likely to trigger different downstream circuitry and underlies the concentration-dependent pheromone perception.

Keywords: vomeronasal organ, olfactory, pheromone, concentration, sex discrimination, masking

Introduction

In terrestrial animals, pheromones play an important role in intra-species communication, eliciting a range of innate and often stereotyped behaviors (Birch, 1974; Wyatt, 2003). Animals have evolved various means, such as urine marking, fecal deposition and flank marking, to broadcast pheromone signals (Ralls, 1971; Humphries et al., 1999; Rich and Hurst, 1999; Hurst and Beynon, 2004). The deposited pheromones can have long lasting effects, especially for the less volatile compounds produced in the vertebrate species (Wyatt, 2003). Moreover, vertebrate animals have evolved strategies to deposit pheromones with carriers, such as the lipacalin family of proteins, to slow down the fading of signals (Keverne, 1998; Beynon and Hurst, 2003). Because pheromones from different individuals are deposited at various times, the concentration and the context in which these signals are detected vary by a large degree and have strong influences on innate behaviors (Bronson, 1979; Kaba et al., 1989; Johnston, 1998; Brennan and Kendrick, 2006). In territorial marking behaviors, for example, high and low concentrations of urinary pheromones have diametric effects (Beauchamp et al., 1982; Nevison et al., 2003; Hurst and Beynon, 2004). Male mice usually avoid fresh, high concentration urine marks of other males, whereas stale or low concentration of urine elicits strong countermarking behaviors (Humphries et al., 1999). On the other hand, high doses of pheromones are generally required to trigger endocrine changes. For example, Drickamer and colleagues reported a dose dependent delay of estrus onset by urine from group-housed female mice (Drickamer, 1982). Similarly, direct contact with male urine is significantly more potent than soiled bedding in accelerating estrus onset (Drickamer and Assmann, 1981; Drickamer, 1986).

It is not understood how pheromone at different concentrations affects innate behaviors. One possibility is that high concentration of pheromones activates more neurons to reach the threshold of behavioral output. In the main olfaction system, increasing odor concentrations leads to the activation of larger numbers of sensory neurons and more glomeruli in the olfactory bulb (Mori et al., 1999; Rubin and Katz, 1999; Meister and Bonhoeffer, 2001). However, it is not clear whether the VNO neurons respond similarly. It has been reported that the pheromone activation of the VNO neurons is highly specific and these neurons are not activated by additional ligands even at high concentrations (Leinders-Zufall et al., 2000a). Alternatively, synergistic and antagonistic interactions among pheromone components with their receptors could produce complex response patterns such that distinct sets of neurons are activated at different concentrations. The VNO expresses ~250 G-protein-coupled, seven-transmembrane receptors (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997; Pantages and Dulac, 2000; Zhang et al., 2004; Yang et al., 2005). Each vomeronasal neuron expresses only one specific type of these receptors. Projections from neurons expressing the same receptor converge to the accessory olfactory bulb in a stereotyped manner (Belluscio et al., 1999; Rodriguez et al., 1999; Del Punta et al., 2002; Wagner et al., 2006). Thus, the differential activation of VNO neurons is likely to trigger neural circuitries that lead to distinct behavioral outputs. In this study, we measured the effective concentration of pheromones reaching the VNO and examined concentration-dependent activation of the VNO.

Materials and Methods

Animals

The generation of the tetO-G-CaMP2 mice was described previously (He et al., 2008). These mice were crossed to OMP-IRES-tTA line (Yu et al., 2004) to restrict expression of G-CaMP2 in the olfactory sensory neurons. Pheromone-evoked responses were obtained from a total of twenty-four 2-6 months old mice (thirteen males and eleven females). Dye labeling experiments were performed using a total of 12 animals of the C57BL/6J strain. Animals were maintained in the Lab Animal Service Facility of Stowers Institute at 12:12 light cycle, and provided with food and water ad libitum. Experimental protocols were approved by the Institutional Animal Care and Use Committee at Stowers Institute and were in compliance with NIH Guide for Care and Use of Animals.

Electro-Vomeronasograms (EVG) Recording

In EVG experiments, local field potentials were recorded from the microvillous layer of intact VNO sensory epithelia as previously described (Leinders-Zufall et al., 2000a; Leypold et al., 2002). Briefly, the VNO epithelium was exposed and perfused with oxygenated mouse artificial cerebrospinal fluid (mACSF). Field potential was recorded using glass pipettes (10 micron diameter) connected to an AI 401 pre-amplifier (Molecular Device). The signals were further amplified by a signal conditioner (Molecular Device), digitized at 1 kHz and low-pass filtered at 20 Hz. The data was further analyzed using Clampfit 10.0 (Molecular Device). Urine samples at dilutions specified in the text were delivered through another glass pipette (10 μm diameter). Urine samples were delivered to the recording sites by a 1-second pulse of air pressure controlled by Pressure System 2 (Toohey Company). Phenol-red at 0.1% was added to all urine and control solutions such that the pulse of application could be visualized. The constant overflow of oxygenated mACSF quickly removed the urine application away from the VNO surface, as visualized from the dispersion of phenol-red. Occasionally, pressure-delivery of urine induced mechanical artifacts. The artifacts were characterized by the abrupt changes in electrical signals timed to the onset and offset of the application. The mechanical artifacts were readily distinguishable from urine-induced responses, which were characterized by significantly slower and smoother time courses. Records with artifacts were excluded from further analyses. Each recording was obtained from a single site with a single application of urine. Recordings were performed with seven mice with 4-7 recording sites used from each VNO preparation. Recordings were first made from a site downstream of the superfused mACSF and moved upward, ending with the most upstream site. This order of recordings precluded potential inactivation or desensitization of neurons that might result from multiple exposures. Traces shown in Figure 1 were those with amplitude close to the mean as shown in 1D. The composition of mACSF is (in mM): 130 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 1.25 NaH2PO4, 25 NaHCO3, 10 Glucose.

Figure 1
Effective concentrations of urine that activate the VNO

Dye labeling of the VNO

Fresh urine was collected and used to dissolve rhodamine 6G (Sigma) to 100 μM. The dye-containing urine was then painted onto the urogenital area of anesthetized female mice. C57BL/6 male mice were then introduced to the cages containing the painted mice one at a time. The investigation of the females by the males was monitored to count the number of investigations. A bout of investigation is defined as a period in which the male continuously poked and pushed into the urogenital area of the female. Disengagement from the area was considered as the end of a bout. Following either a single bout or three bouts of investigation, the male animals were sacrificed and the VNOs were dissected out for confocal imaging. After each individual VNO was taken out from the nasal cavity, the lateral vomer bone encasing the VNO was removed. The blood vessel covering the neuroepithelia was lifted to expose the dendritic surface of the sensory epithelium. The lumenal surface was imaged using the Zeiss LSM 510 confocal system mounted on a Zeiss AxioScope2 FS microscope. A 5X air lens (N.A. = 0.15) was used to perform the imaging. The samples were excited using a 514 nm wavelength argon laser and the emission light was detected by a Meta detector set between 544-576 nm wavelength. Pinhole size was set at 82 μm. Z-series of optical sections were obtained at 10 μm steps. For each region of interest, signals measured at different Z-depth were plotted. The peak amplitude of the signals was used to estimate the dye concentration within the region of interest. Under the same optical condition, Rhodamine-6G solutions at 0, 0.1, 0.5 and 1 μM concentrations were measured. A standard curve based on the free dye measurement was extrapolated and used to estimate dye concentrations in the VNO. Similar procedures were performed for sulforhodamine 101 and rhodamine-conjugated dextran.

To estimate the thickness of the mucus layer, signals from the Z-series measurement for each ROI were deconvolved from the Z-axis point spread function (PSF) of the confocal scope, assuming a Gaussian distribution along Z. The optical section thickness was determined by the pinhole size and equaled 50 μm in these imaging experiments. The deconvolved signals were then fitted with a Gaussian curve and the full width at half maximum (FWHM) of the fitted curve was used to estimate the thickness of the lumenal mucus.

Pheromones and Chemicals

Urine samples were collected from animals using the free-catch method. The freshly collected urine samples were frozen at −80°C until use for up to three months. After thawing the samples, the tubes were spun for 1 min to collect the condensations and reduce the loss of substances. All other chemicals were obtained from Sigma Aldrich. Recombinant ESP1 was prepared from a plasmid provided by Dr. K Touhara as described in Kimoto et al (Kimoto et al., 2005).

VNO Slice Preparation

2-6 months old mice of both sexes were decapitated following CO2 euthanasia. The VNO was removed from the bone capsule and embedded in 4% low melting agarose. 200 μm coronal slices were prepared in oxygenated mASCF at 4°C using Vibratome 3000 sectioning system. Slices were kept in oxygenated (95%O2/5%CO2) mACSF at room temperature for up to eight hours.

Calcium Imaging

Imaging of VNO slices were performed as in (He et al., 2008). VNO slices were continuously perfused with oxygenated mACSF at room temperature at a speed of ~1ml/min. The flow was uni-directional resulting from the placement of the inlet and outlet at opposite ends of the slices. The VNO slices were placed such that the dendrites face the incoming stream. Urine was delivered at various dilutions via BSP-8PG systems using 8, 12 or 16-channel micromanifolds (ALA Scientific). To minimize mechanical artifacts, a continuous flow of Ringer solution (in mM: 115 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 25 NaHCO3, 5 HEPES) was maintained during the experiment. Flow speed was controlled at 2-3 μL per second. Delivery of urine was achieved by the simultaneous opening of a urine valve and the closing of the Ringer solution valve to minimize the change in pressure within the manifold and avoid introducing mechanical disturbance to the slices.

Time-lapse recording of fluorescent signals from the VNO were performed on the Zeiss AxioSkope FS2 microscope with a 10X water-dipping lens (0.3 N.A; 3.3mm working distance). An EXFO X-Cite 120PC light source equipped with a bandpass filter (450-490nm) was used to excite the samples. The epifluorescent images were acquired by a CCD camera (Zeiss HRM) with 2×2 or 4×4 binning depending on the expression levels of G-CaMP2 signal. Most of the data reported here derived from a mouse line designated as 12J, which stably expresses G-CaMP2 in ~50% of the neurons. The same line has been used in our previous publication (He et al., 2008). At the end of some experiments, confocal images were taken to ensure no cells overlapped in the regions of interest. For most experiments, repetitive applications and reverse order applications were performed to examine reproducibility of the responses. To ensure the health of the cells and to reduce potential discrepancies introduced by photo-bleaching, individual slices were recorded for no more than 60 minutes, or 15 recording sessions, each one-minute long. Except for experiments testing urine-induced inactivation, a one-five minute interval was introduced between any pairs of stimuli.

Loose Patch Recordings

Glass electrode (OD 1.2 mm, ID 0.9 mm) was fabricated and polished to have resistance ranging from 3 MΩ to 7 MΩ with P-2000 micropipette puller (Sutter Instruments). Extracellular recordings were performed after forming ~100 MΩ seals onto visually identified cells. The pipette solution contained Ringer solution. Signals were amplified and recorded with MultiClamp 700A amplifier and digitized with Digidata 1440A (Molecular Devices). Spontaneous action potentials and resting membrane potentials were measured under current clamp mode (I = 0) with 10-kHz sampling rate and filtered at 0.5 kHz. Data were acquired and analyzed with Clampex 10.1 and Clampfit 10.1 software (Molecular Devices).

Data analysis

Image processing and data analyses were performed using ImageJ v1.42 software (http://rsb.info.nih.gov/ij/, NIH, Bethesda, MD) as described previously (He et al., 2008). Briefly, a background image sequence was generated by applying Gaussian filter (radius 50 pixels) from the raw image sequence. A scattering-corrected image was obtained by subtracting the background from the raw image sequence. Subsequently, the scattering-corrected image sequence was divided into pre- and post- pheromone application substacks. Responding cells emerged from the background after subtracting the pre- from the post-application stack. To obtain information on response amplitude and dynamics, individual responsive cells were identified manually using the Multi-Measure Plug-In in ImageJ. Response curves were plotted as ΔF/F. To minimize the signals introduced by the auto-fluorescence of urine and light scattering from the responsive cells, randomly selected ROIs that were not neighboring any obviously responsive cell were used to calculate overall background changes.

The response heatmaps were produced using a custom-written program in either Matlab (Mathworks) or Bioconductor R package (http://www.R-project.org). The maps were based on the value according to the ΔF/F of each individual response. Cells responding only to a single urine application were not included in the plot.

To perform PCA and cluster analysis, a response matrix was first generated by assigning 1 to cells with ΔF/F> =0.1 and 0 to cells with ΔF/F<0.1. For hierarchical clustering, the R hclust() function was applied to the response matrix to group the samples. Pairwise Pearson correlation values were calculated between samples and the distance was defined as 1-correlation. The similarity between different samples was plotted as a dendrogram, which grew heuristically by merging pairs of most similar samples into clusters.

Results

Concentration range of natural pheromones detected by the mouse VNO

The VNO is encapsulated in a semi-blind bony structure with one opening connected to the nasal cavity (Wysocki and Lepri, 1991; Keverne, 2002). An active pumping mechanism is required to bring pheromones into the lumen of the VNO (Meredith and O’Connell, 1979; Meredith et al., 1980; Pankevich et al., 2003). The unique mechanism by which pheromones are brought into the VNO, as well as the non-volatile nature of some pheromones, make it difficult to infer the amount of pheromones reaching the VNO directly from their sources.

To estimate the concentration of urine that reached the VNO lumen during free-moving investigation, we painted the urogenital areas of anesthetized animals with mouse urine mixed with different concentrations of a water soluble dye, Rhodamine 6G. Rhodamine 6G is a polar molecule with a molecular weight of 479.02. These properties are similar to what have been characterized for several putative mouse pheromones in the small molecular weight class and are similar to that of small peptides (Novotny et al., 1984; Novotny et al., 1985; Jemiolo et al., 1986; Jemiolo et al., 1989; Novotny et al., 1990; Price and Vandenbergh, 1992; Novotny et al., 1999; Beynon and Hurst, 2003; Nevison et al., 2003). This dye has been used to study the access of non-volatile compounds to the VNO and the main olfactory epithelium (Wysocki et al., 1980; Leinders-Zufall et al., 2004; Spehr et al., 2006). We reasoned that Rhodamine 6G would allow us to estimate the amount of pheromone entering the VNO in a semi-quantitative way, although it might not fully recapitulate the concentration of various pheromones in urine being carried to the VNO.

When male mice were allowed to investigate the painted subjects, fluorescent signals from the dye molecules were observed in their vomeronasal organs (Fig. 1B). To quantify the amount of dye reaching the VNO, we employed laser scanning confocal microscopy to measure the levels of fluorescence in the VNO. We conducted Z-series measurement of fluorescent signals in several randomly selected areas. The signal traces for different regions displayed peak values at different depths (supplemental Fig. 1). By comparing the peak fluorescent intensities of various regions with a series of standard dilutions of the dye, we found that dye concentration inside the VNO lumen reached ~0.5% and ~1.5% of that in urine for the 1- and 3-bout investigations, respectively (Fig. 1B, C). The relative concentration reaching the VNO increased with repeated investigation; 3-bout investigations resulted in approximately three-fold increases in the amount of dye reaching the VNO (Fig. 1 C). We also obtained similar results using a different dye, sulforhodamine 101 (MW 606.71), in both 1-bout and 3-bout experiments (supplemental Figure 2). Interestingly, a higher molecular weight conjugate, rhodamine dextran (MW 10K), was less effective in entering the VNO and reached 0.2% of the dye level in urine in a 3-bout investigation (supplemental Figure 2). This was approximately ten times less than the lower molecular weight dyes and suggested diffusion could play an important role for the pheromones to reach the VNO.

Based on the Z-series measurement of the fluorescent signals, we also estimated the depths of VNO mucus. By deconvolving image signals from the Z-axis point spread function of the confocal scope, we obtained an average of 65.14± 22.92 μm (mean ±s.d.) full width at half maximum (FWHM) value for all the regions measured (supplemental Fig. 3). This number provided an estimate of the mucus thickness.

We next tested the detection limit of natural pheromones by the VNO. We addressed this issue by conducting electro-vomeronasograms (EVG), field potential recordings, from the microvillus surface of the VNO. At 10−6 urine dilution, the variance of the response was comparable to the amplitude, suggesting that the detection threshold was around this concentration (Fig. 1D, E). Stepwise increase in urine concentration up to 10−2 dilution resulted in increases in EVG amplitude. Taken together, these data indicated that the mouse VNO could detect natural pheromones varying in concentration of at least four orders of magnitude, from 10−6 dilution to a few percentages of whole urine.

Patterns of activation by urine at different concentrations

In the next set of experiments, we examined dose-dependent activation of the VNO at the single cell level. We conducted calcium imaging experiments from a large population of neurons in VNO slices prepared from transgenic mice expressing the calcium sensor G-CaMP2 in the vomeronasal neurons (He et al., 2008). Pheromones activate the vomeronasal neurons through the activation of their receptors and elicit an increase in intracellular calcium (Leinders-Zufall et al., 2000a; Leinders-Zufall et al., 2004; He et al., 2008). Our concurrent recordings of calcium signals and extracellular electrical signals showed that calcium signals correlated with urine-evoked spiking activities in the VNO neurons (Fig. 2C). This was in agreement with previous reports (Leinders-Zufall et al., 2000a). Thus, calcium imaging from G-CaMP2 expressing VNO neurons provided readout of pheromone activations. The system also allowed us to visualize large populations of neurons responding to various pheromone stimuli. Due to the mosaicism in transgene expression, G-CaMP2 was stochastically expressed in the neuronal population in the VNO. The stochastic expression pattern allows neurons expressing different receptors to be sampled. In the slice preparations, each slice was estimated to contain approximately 200-300 G-CaMP2 expressing cells. Urine at various concentrations evoked reproducible responses from 40-90 cells per slice, corresponding to ~25-35% of G-CaMP2 positive cells. A Total of 557 cells from nine slices obtained from six animals (four males and two females) responded to different urine samples (supplemental Table 1). Ringer control elicited little responses (supplemental Movies 1 and 3; also see Fig. 4).

Figure 2
Dose-dependent response to mouse urine
Figure 4
Dose-dependent activation of VNO neurons by individual urine samples

When different dilutions of male and female mouse urine were applied, we observed different populations of cells being activated and the responses to different dilutions were consistent across experiments using VNO slices from both male and female animals (Fig. 2A, B; 9 slices from 6 animals; supplemental Table1). In addition to changes in the response amplitude (Fig. 2G, H), we found a general increase in the number of activated cells by high concentration urine from both male and female mice (Fig. 2A, B and D; supplemental Table 1). Urine at 10−2 dilution elicited responses from about five times the number of cells as at 10−6 dilution (n=418 at 10−2 and 81 at 10−6). There was a slight difference between the change in the number of cells activated by male and female urine -- male urine appeared to elicit a sharper increase in activated cells at 10−2 dilution (Fig. 2D). Urine further diluted to 10−8 elicited weak responses from very few cells (supplemental Fig. 4).

Detailed analyses of the responding neurons revealed a dynamic recruitment of different populations of cells at various concentrations. The profiles of cells activated by various concentrations of urine were distinct from one another (Fig. 2E, F and supplemental Table1). Quantitative analysis further revealed that individual cells also displayed different dose-dependent responses. Based on their concentration dependent activation, we grouped the neurons into four broad categories. Type I cells were only activated by low but not high concentration of urine (Fig. 2E, F and G). They constituted about 9 % of all responding cells (n=52 out of 557 total cells; Figure 2I). Type II cells displayed responses across all concentrations tested (Fig. 2E, F and G) and represented 2.7 % (n= 15) of the cells. Type III cells were those activated by high concentrations of urine and represented the largest population (~69 %; n=383) of responding neurons (Fig. 2E, F, H and I). Some of these cells showed canonical dose-response curves, i.e., increased urine concentrations elicited larger response amplitudes that plateaued at higher concentrations (Fig. 2H). Another group, the type IV cells (~19 %; n=107), showed atypical responses. Some of the Type IV cells were activated by urine at both low and high concentrations but not at the intermediate ones, whereas others were activated by intermediate concentrations only (Fig. 2E - I).

The observation of the Type I and Type IV cells was surprising and had not been reported before. In experiments with repeated applications of different urine samples, cells showed reproducible responses to the same stimuli at low or high concentrations of urine (supplemental Figs. 5 and 6). A previous study of VNO response to natural stimuli with multi-electrode recordings largely revealed monotonic increase in response amplitude to increasing concentration of urine from 3×10−4 to 1/30 dilution of urine (Holy et al., 2000). This was largely consistent with the Type III cells, which constituted the majority of responding neurons and were activated at relatively high concentrations of urine (>10−4 dilution). The Type I cells were only revealed at the lower concentrations because they were likely obscured by high concentrations of urine (see below). Therefore, there is no significant contradiction in observations between the two studies.

Because of the presence of different types of cells, the identities of responding cells for low vs. high concentrations were dramatically different. Most cells activated at 10−6 responded also to 10−5 and 10−4 dilutions (Fig. 2E, F and G; Type I). Cells that were activated at 10−3 were mostly activated at 10−2 (Fig. 2E, F and H; Type III). Only 2.7% of the responsive cells were shared by both low (10−6-10−4) and high (10−3-10−2) concentrations of urine (Figs. 2A, B, F and G; Type II). The diminished response from one population of cells and the activation of another implied that the perception of the same urine at various concentrations were likely to be different.

Information conveyed by different concentrations of urine

We wondered whether the patterns of activity could reveal the nature of the signals. Our previous study has shown that urine contains cues to allow the discrimination of gender, strain and individuals (He et al., 2008). In particular, we have found a very small set of neurons that uniquely identify the sex of an animal. The male urine specific cells (MUSCs), responded to all male urine samples regardless of strain, but not to any female urine, and vise versa for the female urine specific cells (FUSCs) ((He et al., 2008). We performed similar analyses across different concentrations of urine samples by stimulating the VNO slices with urine from multiple individuals of strain and sex. These experiments were performed from an additional four slices from three animals (one male and two females). At high concentrations, these cells were readily identifiable and the vast majority of MUSCs and FUSCs were activated at 10−2 dilution (Fig. 3A-C). The percentage of MUSCs and FUSCs dropped significantly at 10−3 dilution. At 10−2, cluster analyses of the patterns of activation by multiple urine samples showed that they were segregated first according to sex, then according to strain, consistent with our previous report (Fig. 4A and B) (He et al., 2008). High concentrations thus allowed the discrimination of individual urine samples according to the genders.

Figure 3
Dose-dependent activation of MUSCs and FUSCs

In contrast, low concentrations of urine-elicited responses from a smaller population of cells, none of which could be identified as MUSCs or FUSCs (Fig. 4C). At 10−4 dilution, the population of cells activated was similar to that activated by 10−5 and 10−6 dilutions (Fig. 2E, 2F). However, cluster analyses showed no pattern of segregation according to sex (Fig. 4D). Nevertheless, these patterns of activation were different for different strains and different individuals, suggesting that even at low concentration natural pheromones contain sufficient information to signal individual differences.

Response to single pheromone compounds

What is the mechanism underlying the dose-dependent activation of distinct cell populations? We wondered how well individual pheromone compounds could recapitulate the concentration-dependent activation by urine. The VNO expresses two large classes of pheromone receptors, the V1Rs and the V2Rs (Dulac and Axel, 1995; Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). Low molecular weight organic compounds most likely activate the V1R family of receptors while the V2Rs are likely to be activated by protein or peptide pheromones (Leinders-Zufall et al., 2000a; Loconto et al., 2003; Leinders-Zufall et al., 2004; Kimoto et al., 2005). We therefore studied the dose-dependent activation of VNO neurons by synthetic compounds that included 2-heptanone, 2,5-dimethylpyrazine (DMP) and α- and β-farnesenes, sulfated steroid compounds (Jemiolo et al., 1986; Jemiolo et al., 1989; Novotny et al., 1990; Nodari et al., 2008), as well as the ESP-1 and MHC peptides (Leinders-Zufall et al., 2004; Kimoto et al., 2005; Leinders-Zufall et al., 2009).

The urinary compounds were found to be present at micro-molar concentrations in mouse urine (Novotny et al., 2007; Nodari et al., 2008).We therefore stimulated VNO slices with single compounds across a range of concentrations (10−13 -10−5 M) that were likely to be encountered by the neurons in natural settings. Similar to urine, increased concentration of single compounds elicited responses from more cells (Fig. 5). For example, DMP, a presumed female pheromone at 10−5 M activated ~6x the number of cells of that at 10−11 M (Figs. 5A and B). The ESP-1 peptide, expressed in the male mice, activated ~8x the number of cells across six orders of concentrations (10−13-10−7 M; Figs. 5D and E; supplemental Movie 4). Detailed examination showed that different cells exhibited sigmoidal dose-response curves (Figs. 5C and 5F). These cells showed distinct sensitivities to the same chemicals, spanning four orders of magnitude. We analyzed more than 200 cells (from 3 male and 3 female animals) responding to single compounds and only found four cells showing non-conventional dose-dependent response.

Figure 5
Dose-dependent responses single pheromones

Masking of Type I cells

What is the mechanism responsible for the non-conventional dose-dependent activation of Type I cells? In the main olfactory neurons, strong excitation can lead to temporary suppression of spikes due to inactivation of the voltage-gated channels (Duchamp-Viret et al., 2003; Rospars et al., 2008; Tan et al., 2010). The vomeronasal neurons, however, do not display this behavior. They fire non-adapting action potentials and the responses show a generally positive correlation with stimulus intensity (Liman and Corey, 1996; Holy et al., 2000; Leinders-Zufall et al., 2000b; Ukhanov et al., 2007; Nodari et al., 2008). Because of this property, it is unlikely that temporary spike suppression can underlie the unconventional responses. Nevertheless, we performed simultaneous electrophysiological recording and calcium imaging to examine the mechanism. Recording from a type I cell showed that a strong calcium response coincided with increased spiking at low concentration of urine (10−5 dilution). The cell fired few spikes and showed little calcium response at higher concentrations (10−4 and 10−3 dilutions) (Fig. 6A). The calcium responses of the type III cell at high concentration of urine (10−2 dilution) also correlated with the spike responses (Fig. 2C).

Figure 6
Masking of low concentration urine by high concentration urine in mixtures

Because individual putative pheromone components displayed conventional dose-dependent response, we reasoned that two likely mechanisms could explain the behavior of Type I cells. The diminution in responses to high concentration pheromones could be due to inactivation or desensitization. However, we observed little inactivation in single compound experiments. Alternatively, these neurons could be actively suppressed by compounds in high concentration urine. We tested these two hypotheses by examining the response of VNO neurons to different urine samples at different concentrations and their mixtures.

We first compared the activation patterns by individual urine samples at 10−4 and 10−2 dilution to that of the mixtures (Fig. 6B-C). Surprisingly, the patterns of activation by these mixtures were not simply the sum of the two samples. Patterns of activation elicited by mixtures generally conformed to that elicited by high concentration urine. The activation by a mixture of 10−2 B6 male urine and 10−4 CD-1 male urine largely recapitulated that of the 10−2 B6 male and vise versa (Fig. 6B). This was not simply due to the smaller number of cells activated by low concentration urine. A number of cells that were activated at 10−4 dilution no longer responded to the mixture. With no exception, all cells responding to low but not high concentrations of urine failed to respond to a mixture if it contained high concentration urine (Fig. 6B and C).

To test the desensitization hypothesis, we next conducted experiments in which low concentration urine was applied immediately after high concentration urine or urine mixtures. If a cell responding to low concentration urine was desensitized at high concentration, one would expect the subsequent application of low concentration urine to elicit smaller or no responses. However, consecutive applications of urine at different concentrations showed little desensitization or inactivation (Fig. 6D). This was consistent with results from a set of experiments examining the response of VNO neurons to paired-pulse application of urine stimuli with various inter-pulse intervals. In these experiments, we observed little sign of inactivation, even with successions of applications with 10-second inter-pulse intervals (supplemental Fig. 5). In another example, the activation of a cell that responded to urine from a CD-1 male at both low (10−4) and high (10−2) concentrations, but not to urine from a B6 male at high concentration (10−2), was suppressed by the mixture containing 10−2 B6 urine (Fig 6C; cell 3). If desensitization was responsible for the loss of activation, we would have expected that the cell not respond to 10−2 CD-1 urine. Moreover, we observed that the masking was dose dependent. For example, we found cells activated by 10−4 B6 but not 10−2 CD-1 urine responded to a mixture of 10−3 CD-1 and 10−4 B6 urine with intermediate amplitude (Fig. 6E). Taken together, these results suggested that the suppression of response at high concentrations was due to the presence of an inhibitor in concentrated urine. The effect is specific because we did not observe such masking effect among urine samples at high concentrations (supplemental Fig. 7). These observations further suggested that the neurons activated at the two urine concentrations were activated by different components in the urine. Because type IV cells are not homogeneous, we currently do not have the means to discern and study them. Nevertheless, their behavior may result from more complex interactions among the pheromones.

Discussion

In the natural environment, innate investigative behaviors result in the exposure of the VNO not only to different concentrations of pheromones, but also to cues mixed from different individuals. Our studies reveal the complex concentration-dependent activation of the VNO. High concentration pheromones activate more cells, most likely due to more pheromone cues reaching the activation threshold. On the other hand, cells activated by low concentration urine are masked by high concentration ones. As a result, urine at low and high concentrations activates distinct subsets of cells, with an apparent transition between 10−4 and 10−3 dilutions. In a previous report, neurons responding differentially to male and female urine were observed across the concentrations tested (Holy et al., 2000). In our experiments, pairwise comparison of VNO activation by individual male or female urine samples indeed shows differential responses, even at low concentrations. However, at low concentrations the MUSCs and FUSCs are not activated. In our previous study (He et al., 2008), we found these two populations of neurons are essential for sex identification. Thus, the differential responses elicited by low concentrations of urine more likely reflect individual differences. Only during active investigations of the urogenital areas can high concentrations of urinary pheromones reach the VNO. At this level, urine can activate the MUSCs and FUSCs and provide unambiguous information that allows fine discrimination of individuals, sex, reproductive and social status. Lower concentrations of pheromones appear inadequate to provide such information.

Strikingly, lower concentrations of urine activate subsets of cells that are distinct from those activated by high concentrations. Moreover, the activation of these cells appears to be suppressed by compounds that are effective in high concentration urine. The masking effect implies that the neurons activated at different concentrations are likely to express different receptors and tuned to different pheromone molecules in the urine. Hence, the downstream neural circuits are likely to be activated differentially by different concentrations of urine and convey separate social signals. Perceptual masking is observed in many sensory systems, but mostly results from central mechanisms that involve network interactions in the brain (Popper and Fay, 1992; Logothetis et al., 1998; Macknik and Livingstone, 1998; Macknik, 2006; Yost et al., 2008). Competitive inputs may result in the suppression of certain signals as in the case of binocular rivalry (Blake and Logothetis, 2002; Zeman, 2004; Tong et al., 2006). In other cases, selective attention may result in the suppression of certain inputs (Tse et al., 2005; Macknik et al., 2008). Perceptual masking in the olfactory system olfactory system appears to be an exception, as some odors can directly mask the activation of the olfactory sensory neurons (OSNs) in the nose (Kurahashi et al., 1994; Duchamp-Viret et al., 2003; Rospars et al., 2008). This is likely due to fact that direct binding of odorants to their receptors are required to activate the OSNs. Masking odorants may bind to the same binding pocket or to a different site to result in competitive or non-competitive antagonism. However, such antagonistic masking has not been observed or proposed for pheromone detection, although suppression was observed in the accessory olfactory bulb (Hendrickson et al., 2008). Unlike the main olfactory neurons, which generally are more promiscuous in interacting with different odorants, pheromone-sensing neurons are highly selective (Buck, 2000; Leinders-Zufall et al., 2000a; Wyatt, 2003). The selectivity presumably allows highly specific information to be conveyed among animals of the same species (Wyatt, 2003). Our observation that pheromones could mask each other thus offers insight into a novel means to control the information being transmitted among animals.

At this time, we do not know the precise nature of the signals conveyed by the low concentration urine, nor do we know the behavioral implications of the suppression of these signals. Considering that pheromones deposited in urine markings are likely to reach the mouse VNO at lower concentrations, we speculate that cells activated at these concentrations could be used to obtain a snapshot of the presence of other animals in the surroundings. For example, both male and female mice countermark over urine deposits left by other mice (Rich and Hurst, 1998, 1999; Nevison et al., 2003; Hurst and Beynon, 2004; Brennan and Kendrick, 2006). The freshly deposited urine could serve to mask the marks left by other animals and the masking of low concentration pheromones may reduce the influence of unwanted signals. This is important for forming pheromone-related memory as the animals live in an environment laden with signals from many individuals. Additionally, volatile compounds in urine marks will get weaker over time whereas peptides may get concentrated. The differential patterns evoked by marks at different ages can provide further information of scent marks. In any case, behavioral experiments have shown complex interactions among different sources of pheromones. The presence of female pheromones could significantly dampen inter-male aggression whereas male pheromones override signals from group-housed female in synchronizing estrus (Bronson, 1971). Urine from sexually-experienced male can also suppress the effect of female urine in elevating luteinizing hormone levels in conspecific males (Clancy et al., 1988) Our results with urine mixtures suggest some antagonistic interactions at the receptor level although computations within the neural circuitry in the brain could also occur.

Supplementary Material

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ACKNOWLEDGEMENTS

We thank Drs. W. Wiegraebe and D. Zhu for help on imaging and statistical analyses. We thank E. Gillespie, P. Zelalem, Megan Fracol, G. Hattem, M. Elmore, S. Klinefelter, K. Cavanaugh, Lab Animal Service Facility and Microscopy Center at Stowers Institute for technical assistance. The ESP1 plasmid was generously provided by Dr. K. Touhara from University of Tokyo. We also thank Drs. M. Gibson, R. Krumlauf, H.Y. Mak and K. Si for thoughtful discussions and critical reading of the manuscript. This work is supported by funding from Stowers Institute and the NIH (NIDCD 008003) to CRY. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Deafness and Other Communication Disorders or the National Institutes of Health. U.S. patent pending for the tetO-G-CaMP2 mice for Stowers Institute, CRY and LM.

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