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. Author manuscript; available in PMC: 2016 May 18.
Published in final edited form as: Curr Biol. 2015 Apr 30;25(10):1340–1346. doi: 10.1016/j.cub.2015.03.026

Innate Predator Odor Aversion Driven by Parallel Olfactory Subsystems that Converge in the Ventromedial Hypothalamus

Anabel Pérez-Gómez 1, Katherin Bleymehl 1, Benjamin Stein 1, Martina Pyrski 1, Lutz Birnbaumer 2,4, Steven D Munger 3, Trese Leinders-Zufall 1,5, Frank Zufall 1,5, Pablo Chamero 1,5
PMCID: PMC4439360  NIHMSID: NIHMS674492  PMID: 25936549

Summary

The existence of innate predator aversion evoked by predator-derived chemostimuli called kairomones offers a strong selective advantage for potential prey animals. However, it is unclear how chemically-diverse kairomones can elicit similar avoidance behaviors. Using a combination of behavioral analyses and single-cell Ca2+ imaging in wild-type and gene-targeted mice, we show that innate predator-evoked avoidance is driven by parallel, non-redundant processing of volatile and nonvolatile kairomones through the activation of multiple olfactory subsystems including the Grueneberg ganglion, the vomeronasal organ, and chemosensory neurons within the main olfactory epithelium. Perturbation of chemosensory responses in specific subsystems through disruption of genes encoding key sensory transduction proteins (Cnga3, Gnao1) or by surgical axotomy abolished avoidance behaviors and/or cellular Ca2+ responses to different predator odors. Stimulation of these different subsystems resulted in the activation of widely distributed target regions in the olfactory bulb, as assessed by c-Fos expression. However, in each case this c-Fos increase was observed within the same subnuclei of the medial amygdala and ventromedial hypothalamus, regions implicated in fear, anxiety and defensive behaviors. Thus, the mammalian olfactory system has evolved multiple, parallel mechanisms for kairomone detection that converge in the brain to facilitate a common behavioral response. Our findings provide significant insights into the genetic substrates and circuit logic of predator-driven, innate aversion and may serve as a valuable model for studying instinctive fear [1] and human emotional and panic disorders [2, 3].

Results and Discussion

The mammalian olfactory system is composed of a variety of subsystems that rely on distinct sensory neuron subpopulations for chemodetection [4–7]. Three of these sensory neuron groups–the vomeronasal organ (VNO) [8–10], the Grueneberg ganglion (GG) [11] and subsets of sensory neurons within the main olfactory epithelium (MOE) that express trace amine-associated receptors (Taars) [7, 12, 13]–have been implicated in predator odor aversion through their detection of kairomones, which are semiochemicals released by one species and that benefit a member of another species [14]. To better understand how instinctive behaviors are initiated and controlled by these subsystems, how odor-driven activity from each subsystem is represented and integrated by higher structures in the central nervous system (CNS), and what functional contributions each subsystem makes to social behaviors, we focused on a functional dissection of innate kairomone aversion in mice.

Defensive Behaviors Evoked by Different Predator Odors

Naïve – not previously exposed – wild-type male mice (C57BL/6, denoted as B6) were presented a range of predator-derived odors (Figure 1A) and behavior was monitored automatically (see Experimental Procedures available online). We systematically compared several predator odors whose nasal detection has been associated with distinct olfactory substructures: TMT (2,5-dihydro-2,4,5-trimethylthiazole), which is present in fox feces and activates both the MOE and GG [11, 15]; PEA (β-phenylethylamine), which is found in carnivore urine and activates Taar4 neurons of the MOE [12, 13]; cat fur odor (CFO), which activates the VNO [9, 10]; and 2-PT (2-propylthietane), a GG activator from the stoat anal gland [11] (Figure 1B). Objects impregnated with each stimulus were presented in single trials and behavior was quantified as time spent in the vicinity of the stimulus (Figure 1C), average location in the test cage (Figure1D and 1E), and accumulated distance walked (Figure 1F). All four predator odors elicited robust avoidance in B6 mice (Figure 1C–G). Additionally, some stimuli also produced other types of defensive behaviors. For example, CFO but not TMT, PEA, or 2-PT induced an elevated number of risk-assessment episodes (Figure 1H), characterized by an investigative extended body posture [16]. TMT but not CFO, PEA, or 2-PT caused the mice to bury the stimulus in the cage bedding (Figure 1I). Therefore, all four stimuli evoke a coherent behavioral output, innate predator odor aversion. Other forms of defensive behavior, such as risk assessment and object burying, are displayed only in response to select stimuli.

Figure 1. Comparing Innate Defensive Behaviors Driven by Different Predator Odors.

Figure 1

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(A) Example of a video frame indicating the behavioral assay in which a male mouse is exposed to a cotton pad impregnated with a particular predator odor (object). Time spent in area 1 or 2 was quantified.

(B) Schematic showing proposed olfactory subsystem specificity of the predator odors tested. MOE, main olfactory epithelium; GG, Grueneberg ganglion; VNO, vomeronasal organ; 2-PT, 2-propylthietane ; TMT, 2,5-dihydro-2,4,5-trimethylthiazole; PEA, β-phenylethylamine; Taar4, trace amine-associated receptor 4-expressing neurons; CFO, cat fur odor.

(C) Average time spent by B6 mice in each of the opposing cage areas as depicted in (A). Object was placed in area 1 (n = 13 – 24 B6 mice for each odor exposure). Phosphate buffered saline (PBS) served as neutral control. Area 1: ANOVA: F4,77 = 4.73; p < 0.005; LSD: * p < 0.05; ** p < 0.01; *** p < 0.001; Area 2: ANOVA: F4,77 = 4.15; p < 0.005; LSD: * p < 0.05; ** p < 0.01; *** p < 0.001.

(D) Trajectory plots of the position of a representative B6 mouse (left panel, single B6 mouse) and corresponding heat map graphs of all tested mice (right panel; B6 total) to 15-min exposures of predator odor (blue circle: object position). The black squares in the heat map graphs represent the average position of the animals in the cage.

(E) Average position of B6 mice in the test cage during the 15-min assay. Cages were 30 cm long and objects were placed approximately 5 cm from the edge of the cage. ANOVA: F4,77 = 4.27; p < 0.005; LSD: * p < 0.05.

(F) Average accumulated distance walked during a 15-min exposure to different predator odors (n = 13 – 24 B6 mice for each predator odor).

(G) Average response to a given stimulus as quantified by the aversion index. For each stimulus and mouse, we defined a performance index (see Experimental Procedures), such that avoidance is indicated by a negative value and attraction by a positive one. ANOVA: F4,74 = 4.24, LSD: *p < 0.05.

(H) Number of risk assessment episodes displayed during exposure to different predator odors. CFO but not TMT, PEA, or 2-PT evoked a significant increase in the number of risk assessment episodes. ANOVA: F4,65 = 18.21, LSD: ***p < 0.001.

(I) Percentage of animals that bury the object in the cage bedding. TMT but not CFO, PEA, or 2-PT evoked defensive burying behavior. ANOVA: F4,65 = 5.93, LSD: **p < 0.005 (n = 11–18). Results are presented as means ± SEM.

Kairomones Engage Gαo-dependent and -independent Pathways

To dissect the peripheral neural pathways necessary for innate predator odor aversion, we analyzed behavioral and cellular responses of mutant mice deficient for specific signal transduction components. We first used a mutant strain (cGαo−/− mice) in which the Gnao1 gene (encoding the G protein Gαo) has been conditionally deleted under the control of the Omp (olfactory marker protein) gene [17, 18]. Gαo is essential for sensory transduction in VSNs of the basal layer of the vomeronasal sensory epithelium [17], which detect nonvolatile peptides and major urinary proteins (MUPs) [19]. In behavioral studies, CFO avoidance was abolished in cGαo−/− mice (Figure 2A and S1G), while CFO-induced risk assessment was significantly reduced compared to cGαo+/− controls (Figure S1H). By contrast, the avoidance evoked by TMT, PEA, and 2-PT in cGαo−/− mice was indistinguishable from controls (Figure 2A), indicating that these three stimuli engage Gαo-independent signal transduction mechanisms. Avoidance of the candidate kairomone protein Feld4, a MUP-like orthologue present in cat saliva that elicits avoidance behavior in mice in a Trpc2-dependent manner [10], was similarly abolished in cGαo−/− mice (Figure 2A, Figure S1A–F). Ratiometric Ca2+ imaging of freshly dissociated VSNs [17, 20, 21] showed that both CFO and Feld4 (500 nM) activated ~ 2% of cells screened in either B6 and cGαo+/− controls (n = 8000 – 12,000 cells; Figure S1J, K); a large fraction of cells activated by CFO (47%) were also activated by rFeld4 (Figure S1J, K). By contrast, VSNs from cGαo−/− mice revealed a drastic reduction (p < 0.001) in the responses to rFeld4, comparable to background (control) activity levels and consistent with the idea that this protein is only detected by basal VSNs (Figure S1J, K). Interestingly, cGαo−/− mice also displayed some attraction to CFO (Figure 2A), indicating that they are not anosmic for this stimulus. This emergent behavior likely results from responses to attractive components of this complex stimulus that are unmasked once responses to aversive components are eliminated. Indeed, a fraction of Gαo−/− cells showed responses to CFO at levels ~50% of those seen in controls (p < 0.001), but there was no overlap between CFO-induced activity and background responses obtained with rFeld4 in Gαo−/− cells (not shown). Additionally, cGαo−/− mice showed no attraction to Feld4 (Figure 2A). Therefore, CFO is detected by both Gαo-dependent and –independent vomeronasal transduction mechanisms whereas rFeld4 detection depends solely on Gαo-dependent sensing. Despite a loss of sensory function in the basal VSNs, the VNO of cGαo−/− mice still retains the ability to detect at least some CFO components (e.g. by apical VSNs that do not require Gαo for transduction) even though this activity is not sufficient to drive innate avoidance behavior.

Figure 2. Genetic Dissection of Innate Aversion.

Figure 2

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(A) Aversion index of cGαo−/− (black) vs. cGαo+/− (grey) mice exposed to control (ctrl), TMT, CFO, PEA, 2-PT, or rFeld4. The avoidance to CFO and rFeld4 depends on Gαo. ANOVA: F1,100 = 5.29, LSD: * p < 0.05, **p < 0.01, ***p < 0.001, ns (not significant), p = 0.79 (n = 6 – 21).

(B) Aversion index of Cnga3−/− vs. Cnga3+/− mice exposed to the set of predator odors, demonstrating that the avoidance to 2-PT requires Cnga3 whereas the avoidance to TMT, CFO, or PEA does not. ANOVA: F1,129 = 2.33, LSD: **p < 0.01 (n = 13 mice/genotype).

(C) Aversion index for sham-operated and GGX mice exposed to the set of predator odors. 2-way ANOVA: F1,99 = 2.26, LSD: * p < 0.05, **p < 0.01; ns (not significant), p = 0.08 (TMT-sham vs. TMT-GGX) and p = 0.4 (GGX-control vs. GGX-TMT) (n = 10 mice/condition).

(D) OMP immunofluorescence performed on GG histological sections four weeks after axotomy (GGX; bottom) and in sham-operated animals (top). Green OMP fluorescence is detected in intact GGNs. Scale bar, 50 µm. Results are presented as means ± SEM.

Cnga3 Null Mice Enable Dissection of Innate Kairomone Aversion by the Grueneberg Ganglion

We performed further experiments using OMP-GFP/Cnga3 mutant mice (Cnga3−/− or Cnga3+/− mice) [22]. These mice lack the CNG channel subunit Cnga3 [23–25] and express green fluorescent protein (GFP) in all OMP-expressing cells. Behavioral analyses in Cnga3+/− vs. Cnga3−/− mice revealed that the avoidance to 2-PT was nearly eliminated in Cnga3−/− mice (p < 0.01) whereas the aversion evoked by TMT, CFO and PEA remained normal (Figure 2B and S2A–C). Cnga3 is expressed in both the GG [26, 27] and in small subsets of sensory neurons in the MOE and the septal organ that also express the guanylyl cyclase GC-D (~ 0.1% of OSNs) [23, 28, 29]. Surgical axotomy of the GG (GGX, Figure 2D) eliminated the innate avoidance to 2-PT (p < 0.01) but not that to CFO or PEA (Figure 2C and S2D–F), indicating that 2-PT-evoked avoidance is driven solely by the GG. Although GGX mice showed a trend towards reduced aversion to TMT, this difference was not significant compared to controls (Figure 2C; LSD: p = 0.085). Interestingly, GGX but not Cnga3−/− mice failed to bury objects impregnated with TMT (Figures S2C and S2F), indicating that TMT-evoked object burying is also driven by the GG. However, a functional Cnga3 channel was not required for this effect (Figure S2C). Time-resolved, cellular analyses using ratiometric Ca2+ imaging on acute GG tissue slices obtained from OMP-GFP+/, Cnga3+/− and Cnga3−/− mice [22] in response to chemostimulation supported this division (Figure S2G–K). We identified two major subpopulations of GGNs in OMP-GFP+/− and Cnga3+/− mice: one (nearly 50% of the cells, type 1) that detects 2-PT but not TMT, and another (about 25% of the cells, type 2) that senses TMT but not 2-PT (Figures S2I and S2J). Importantly, while the responses of Cnga3−/− GGNs to 2-PT were nearly absent, those to TMT were similar to controls regardless of the parameter analyzed (e.g. number of responding cells or size of the Ca2+ responses) (Figures S2J and S2K). Therefore, Cnga3 is essential for chemosensory transduction in a subset of GGNs that detect the predator odor 2-PT but not TMT and these neurons are likely required for driving innate aversion to 2-PT. By contrast, GGN detection of TMT is largely independent of Cnga3.

Segregated Kairomone Representation in the Olfactory Bulb

Next, we mapped neural activation in the olfactory bulb following kairomone exposure using immunodetection of the immediate early gene c-Fos. In the accessory olfactory bulb (AOB), we observed a significant increase in the number of c-Fos+ nuclei in B6 mice exposed to either CFO or rFeld4, but not to TMT, 2-PT or PEA (Figure S3A–C). Consistent with results from our Ca2+-imaging experiments, CFO-evoked c-Fos was found in both the anterior and posterior AOB whereas rFeld4 exposure only activated the posterior AOB (Figure S3A–C). In cGαo−/− mice, CFO-induced c-Fos was detected in the anterior but not posterior AOB (Figure S3D, E). Therefore, CFO induces activation of the AOB in both Gαo+ (posterior) and Gαi2+ (anterior) zones, but the activity of the Gαi2+ zone alone is not sufficient to drive innate avoidance behavior.

The necklace glomerulus region of the MOB is innervated by GGNs and GC-D+ OSNs, both of which express phosphodiesterase 2A (PDE2A) in their axons (Figure S3F). We observed a 6- to 8-fold increase in c-Fos+ cells in the immediate vicinity of PDE2A+ glomeruli of 2-PT- and TMT-exposed B6 mice (Figure S3G, H). 2-PT failed to induce any increase in c-Fos+ cells in Cnga3−/− mice whereas TMT caused elevated (~ 7-fold) c-Fos activation in PDE2A+ glomeruli of both genotypes (Figure S3G, H). Similarly, the 2-PT-evoked increase in c-Fos+ cells was abolished whereas the TMT-evoked activity was only partially reduced in GGX mice (Figure S3G, H). Thus, 2-PT-evoked activity in the caudal OB depends entirely on GGN activation and requires a functional Cnga3 channel. TMT-evoked activation of the necklace system does not require Cnga3 and is only partially reduced by GG axotomy, suggesting that these necklace glomeruli receive additional sensory input from TMT-detecting neurons located outside the GG, most likely in the MOE [15, 30]. Some OB glomeruli outside the necklace system are also activated by TMT ([31] and data not shown). Combined with the observation that PEA activates a distinct set of glomeruli in the dorsocaudal OB [32, 33], these findings demonstrate that the representation of predator odors in the OB remains highly distributed across the olfactory periphery and olfactory bulb.

Kairomone Information Converges in the Medial Amygdala (MeA) and Ventromedial Hypothalamus (VMH)

We next asked if these pathways converge at higher levels of the CNS. Exposure of B6 mice to CFO (VNO), rFeld4 (VNO), PEA (MOE) and 2-PT (GG) induced a 2 to 5-fold increase in the number of c-Fos+ cells in the posteroventral MeA (MePV) but not in the posterodorsal MeA (MePD) (Figure3A and 3B). These results suggest that this predator odor information must ultimately all target the MePV (see also [9]). By contrast, TMT failed to induce any significant increase in c-Fos in the MeA (Figure3A and 3B; p = 0.322), consistent with previous mapping of TMT-dependent neuronal activation [31, 34, 35].

Figure 3. The Medial Amygdala, Posteroventral Division, is Activated by Predator Signals Originating from the Grueneberg Ganglion, the VNO, and the Taar System.

Figure 3

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(A) Examples of c-Fos activation on coronal sections through MeA of B6 mice exposed to various predator odors. MePD, posterodorsal medial amygdala; MePV, posteroventral medial amygdala. c-Fos+ nuclei were counted blindly for each genotype or treatment. Images (Bregma −1.46 mm) are displayed as color-inverted (negative) of original fluorescent B/W-acquired images. Scale bars, 100 µm.

(B) Quantification of c-Fos+ nuclei of B6 mice showing that MePV but not MePD is activated by CFO, PEA, 2-PT, and rFeld4. The effect of TMT was not significantly different from control.

(C–E) Quantification of c-Fos+ nuclei in the MePV of cGαo+/− and cGαo−/− mice (C), Cnga3+/− and Cnga3−/− mice (D), and 5 GGX mice (E). B6: ANOVA: F5,36 = 3.39 – 4.64; cGαo: ANOVA: F1,24 = 10.65; Cnga3: ANOVA: F1,23 = 14.11; LSD: *p < 0.05; ***p < 0.001; ns, not significant. GGX: t-test: p = 0.98. N = 5 – 8 mice per stimulus and genotype. Results are presented as means ± SEM.

Our c-Fos mapping also revealed robust activation in response to PEA, 2-PT, CFO, and rFeld4 in a major MePV target region, the dorsomedial subdivision of the ventromedial hypothalamus (VMHdm) [36] (Figures 4A and 4E); the central (VMHc) and ventrolateral (VMHvl) aspects of the VMH lacked activation by any of these cues. CFO failed to elicit any increase in c-Fos+ cells in both the MeA (Figures 3C and S4A) and VMH (Figures 4B and 4F) of cGαo−/− mice. Similarly, the c-Fos increase to 2-PT was eliminated in the MeA (Figures 3D,E and S4B,C) and VMH (Figures 4C,D and 4G,H) of both Cnga3−/− and GGX mice. Thus, the activity we observe in MeA and VMH in response to CFO and 2-PT requires intact function of Gαo in basal VSNs and of Cnga3 in the GG, respectively. Consistent with the results in the MeA, TMT did not induce a significant increase (p = 0.921) of c-Fos+ cells in any of the VMH regions (Figures 4A and 4E).

Figure 4. Cells in the VMH, Dorsomedial Division, Receive Sensory Input from Parallel Olfactory Subsystems Driving Innate Predator Odor Avoidance.

Figure 4

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(A–D) Examples of c-Fos activity in sections through the VMH of B6 (A), cGαo−/− (B), Cnga3−/− (C) and GGX (D) mice exposed to predator odors. Images (Bregma −1.46 mm) are displayed as color-inverted (negative) of original fluorescent B/W-acquired images. dm, dorsomedial VMH; c, central VMH; vl, ventrolateral VMH. Scale bars, 100 µm.

(E) Quantification of c-Fos+ nuclei in the three VMH subdivisions indicates that VMHdm is activated by CFO, PEA, 2-PT, and rFeld4 but not by TMT. ANOVA: F5,34 = 2.63–7.29.

(F–H) Quantification of c-Fos+ nuclei in VMHdm of cGαo+/− and cGαo−/− mice (F), Cnga3+/− and Cnga3−/− mice (G), and GGX mice (H). cGαo: ANOVA: F1,21 = 15.72; Cnga3: ANOVA: F3,23 = 31.7; LSD: *p < 0.05; ***p < 0.001; ns, not significant. GGX: t-test: p = 0.98. N = 5 – 8 mice per stimulus and genotype. Results are presented as means ± SEM.

(I) Scheme summarizing the results. In the context of innate predator odor avoidance, olfactory inputs from three different olfactory subsystems (VNO, GG, Taar system) are processed by separate, parallel neural pathways that provide converging sensory input to MePV and VMHdm. TG, Taar-glomeruli; NG, necklace glomeruli.

Conclusions

Our results indicate that the representation of diverse predator odors – initially widely distributed across the olfactory system – eventually becomes highly organized and spatially confined to specific subregions of the MeA and VMH (Figure 4I). In particular, our observations indicate a central role of VMHdm in the control of innate kairomone aversion. Our results are consistent with studies implicating the MePV in the control of fear and anxiety responses [37–39] and the VMH in defensive, escape, avoidance, and panic-like behaviors in rodents [36, 40–47] and panic attacks in humans [3]. We conclude that the coding strategies underlying kairomone-mediated aversive behavior include (i) the engagement of multiple, distinct olfactory subsystems; (ii) that each subsystem functions in parallel and relatively independently to trigger innate avoidance in a non-redundant manner; and (iii) that these olfactory subsystems generate converging sensory inputs to the same higher-order subnuclei of the CNS. An obvious advantage of these coding strategies in kairomone-mediated avoidance is that an animal receiving these signals would increase its chance of survival by enhancing receptor space to maximize the probability that a life-threatening predator is detected in a timely manner.

Supplementary Material

suppl

Highlights.

  • Individual predator kairomones activate distinct olfactory subsystems.

  • These diverse subsystems can each mediate innate aversion to select kairomones.

  • Kairomone information converges in medial amygdala and ventromedial hypothalamus.

Acknowledgments

We thank Peter Mombaerts for supplying OMP-GFP and OMP-Cre mice, Martin Biel for providing Cnga3 null mice, and Lisa Stowers for a gift of Feld4 plasmid. This work was supported by Deutsche Forschungsgemeinschaft grants CH 920/2-1 (P.C.), Sonderforschungsbereich 894 projects A16 (T.L.-Z.) and A17 (F.Z.), and International Graduate School GK 1326 (K.B.), National Institute on Deafness and Other Communication Disorders grant DC005633 (S.D.M. and F.Z.), a University of Saarland HOMFORexzellent grant (P.C.), the Intramural Research Program of the NIH to L.B. (Project Z01 ES-101643), and the Volkswagen Foundation (T.L.-Z.). Also T.L.-Z. is a Lichtenberg Professor of the Volkswagen Foundation.

Footnotes

Supplemental Information

Supplemental Information includes Supplemental Experimental Procedures and four figures.

Author Contributions

A.P.G., T.L.-Z., F.Z., and P.C. designed and initiated the study. A.P.G. performed all behavioral experiments. B.S. and K.B. performed Ca2+-imaging in VSNs and GGNs. A.P.G. and M.P. performed immunohistochemistry and M.P. performed all lesions. A.P.G., K.B., B.S., M.P, T.L.-Z., and P.C. analyzed data. L.B. provided mouse models. S.D.M., T.L.-Z., F.Z. and P.C. wrote the manuscript and all authors edited the manuscript.

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