Large-scale forward genetics screening identifies Trpa1 as a chemosensor for predator odor-evoked innate fear behaviors

Innate behaviors are genetically encoded, but their underlying molecular mechanisms remain largely unknown. Predator odor 2,4,5-trimethyl-3-thiazoline (TMT) and its potent analog 2-methyl-2-thiazoline (2MT) are believed to activate specific odorant receptors to elicit innate fear/defensive behaviors in naive mice. Here, we conduct a large-scale recessive genetics screen of ethylnitrosourea (ENU)-mutagenized mice. We find that loss of Trpa1, a pungency/irritancy receptor, diminishes TMT/2MT and snake skin-evoked innate fear/defensive responses. Accordingly, Trpa1−/− mice fail to effectively activate known fear/stress brain centers upon 2MT exposure, despite their apparent ability to smell and learn to fear 2MT. Moreover, Trpa1 acts as a chemosensor for 2MT/TMT and Trpa1-expressing trigeminal ganglion neurons contribute critically to 2MT-evoked freezing. Our results indicate that Trpa1-mediated nociception plays a crucial role in predator odor-evoked innate fear/defensive behaviors. The work establishes the first forward genetics screen to uncover the molecular mechanism of innate fear, a basic emotion and evolutionarily conserved survival mechanism.

1) The authors convincingly show that innate freezing triggered by TMT/2MT depends on TRPA1 receptors. However, there is a debate around the validity of TMT as a predator odor. Although it is interesting that innate freezing to this odorant depends on TRPA1 receptors, it would be great to know whether freezing triggered by natural predator odors taps onto the same mechanism. To address this issue the authors could perform a simple experiment testing trpa1 mutant mice to natural predator odors, such as fox feces (from which TMT was isolated) or cat odor. Even if freezing is not reliability triggered by these odors in WT mice, they can look at avoidance. Disruption of innate defensive responses to natural predator odors in trpa1 KO mice would strengthen their argument that trpa1 mediates chemical detection of predators. If TRPA1 receptors are not required for defensive responses, freezing or avoidance, to predator odors, it is still interesting.
2) The authors demonstrate that trpa1 mutants still detect 2-MT, showing c-fos expression in the olfactory bulb and intact conditioned freezing to 2MT after pairings with footshock. However, it is unclear which part of the bulb is activated by 2MT. Given that previous work from the Sakano lab (including one of the co-authors of this study) has shown that the dorsal portion of the olfactory bulb is involved in innate responses to TMT, it would be interesting to know whether KO mice for TRAP1 show normal responses to 2MT. C ould TRPA1 receptors be important for responses in the dorsal portion of the bulb? The authors have the data. Answering this question only requires analyzing c-fos expression in the dorsal portion of the olfactory bulb.
3) The authors should report their statistical analysis more clearly and include a section in the methods about it. This is particularly relevant given that in a few cases it is unclear whether the statistical tests are appropriate for the data. For example for the data in figure 4c, the authors report a one-way ANOVA for an n of 3. Not only the sample size is too small for an ANOVA, but it is unclear what is exactly the data the authors are trying to explain since they show 2 data sets for each receptor (one for TMT and another for 2MT). Is the data being pool? How? When multiple comparisons are shown (* symbols in figures) but a single value of an ANOVA is reported it is unclear which posthoc tests were used and if in all cases correction for multiple comparisons was used.
4) The authors only tested the rescue of trpa1 in trigeminal neurons for responses to 2MT, the most pungent stimulus. I believe that ideally the authors should have tested rescue to TMT or 2MT low dose. Given that repeating that experiment is not trivial the authors should mention the possibility that trpa1 in other tissues would mediate the response to TMT or low dose 2MT, such that the rescue only in trigeminal neurons would not work. The authors mention a possible role of trpa1 in other tissues, but not directly regarding the rescue experiment. This is relevant in light of the general discussion in the field regarding predator versus pungent odors driving innate freezing.
5) The double staining images shown for the rescue experiment (Fig7 G and H) are not very helpful without a quantification of double-labeled cells.
Reviewer #2 (Remarks to the Author): This interesting study proposes a novel unconventional model for the signaling pathways controlling fear-like responses in mice to predator odors that involves the trigeminal ganglia and TRPA1 ion channel. The study attacks a long-standing controversy about the real role of TMT in mice: a fearinducing chemosignal or just as an aversive pungent chemical. Three major questions arise from the results: 1) is TRPA1 a sensor for TMT/2MT? 2) are Trpa1-/-mice still able to detect TMT/2MT? 3) are TRPA1+ cells in the TG driving fear-like responses?
1. The first question seems partially resolved, at least in HEK cells. However, increase of c-fos expression after 2MT exposure was detected in only a fraction of TRPA1+ neurons on the TG and many of the activated TG neurons lacked Trpa1 expression (fig 6f), rising concerns about sensitivity and specificity. More evidence showing that Trpa1 neurons indeed detect TMT/2MT and their level of specificity would strengthen the conclusions. Using a more physiological model or in vivo recordings would address this point, perhaps including other Trpa1-expressing neurons such as DRG. It would also help substantially to use an experimental approach with a better temporal resolution than c-fos staining (C a-imaging or electrophysiology recordings, for example). These experiments should clarify the specificity of TMT/2MT for Trpa1-neurons comparing to potential responses in KO controls.
2. Results using the learned fear assay (Fig 5e and suppl fig 8c,d) indicate that Trpa1-/-mice are indeed able to detect 2MT, likely via the olfactory system (Fig 5c,d). Unfortunately, no data on heterozygous or WT controls in the learned fear assay was included for comparison with the KO. This is an important control to exclude any possible reduction in olfactory performance in Trpa1-/-mice. Reduced olfaction may have an important impact on the behavior and thus, conclusions would need to be reformulated. The cookie test shown on suppl fig 8a is clearly insufficient to evaluate olfaction in Trpa1-/-. A habituation-deshabituation test using low 2MT dose (as on Fig 3) might be more appropriate in this case. In this context, showing that other odorants not detected by Trpa1 (such as those used in refs 17, 47, 48) can induce freezing would greatly strengthen the work.
3. Whether Trpa1+ cells in the TG are driving the observed fear-like responses is unclear to me. First, the 2MT effect on behavior seems to be partial: background (no odor) freezing rates in WT mice ranges 0-20% (Fig 1a) whereas in 2MT-induced in Trpa1 mutants is 2-fold higher, up to 20-40% (figs 2b and 3a). This would be consistent with decreased olfaction. A no odor control in Trpa1-/-mice may clarify this point. Second, authors use a (>2s) decrease in locomotor activity as a proxy for freezing, which is assumed as a fear response. C areful observation of the movies reveals that TMT/2MT not only elicits freezing, but also sitting in distant areas from the stimulus, which can be also interpreted as avoidance. This point should be mentioned in the discussion, which now is overly oriented towards emotional fear. The "emotional" component of aversion is supposed to be lower than fear, fitting better with the classical function of TG neurons. Third, and related with the previous point, it has been reported (in ref. 13) that a subset of olfactory-activated neurons from the cortical amygdala plays a critical role in the generation of innate TMT-driven aversive behavior. Activity in this brain region in Reviewer #1 (Remarks to the Author): 1) The authors convincingly show that innate freezing triggered by TMT/2MT depends on TRPA1 receptors. However, there is a debate around the validity of TMT as a predator odor. Although it is interesting that innate freezing to this odorant depends on TRPA1 receptors, it would be great to know whether freezing triggered by natural predator odors taps onto the same mechanism. To address this issue the authors could perform a simple experiment testing trpa1 mutant mice to natural predator odors, such as fox feces (from which TMT was isolated) or cat odor. Even if freezing is not reliability triggered by these odors in WT mice, they can look at avoidance. Disruption of innate defensive responses to natural predator odors in trpa1 KO mice would strengthen their argument that trpa1 mediates chemical detection of predators. If TRPA1 receptors are not required for defensive responses, freezing or avoidance, to predator odors, it is still interesting. RE: We thank the reviewer for this excellent suggestion. We successfully showed that Trpa1 -/mice were defective for snake skin-evoked innate freezing, avoidance, flight, and risk assessment behaviors as compared to Trpa1 +/mice (Fig. 3f,g). This critical result establishes that TRPA1 mediates natural predator odor-evoked innate fear/defensive responses.
2) The authors demonstrate that trpa1 mutants still detect 2-MT, showing c-fos expression in the olfactory bulb and intact conditioned freezing to 2MT after pairings with footshock. However, it is unclear which part of the bulb is activated by 2MT. Given that previous work from the Sakano lab (including one of the co-authors of this study) has shown that the dorsal portion of the olfactory bulb is involved in innate responses to TMT, it would be interesting to know whether KO mice for TRAP1 show normal responses to 2MT. Could TRPA1 receptors be important for responses in the dorsal portion of the bulb? The authors have the data. Answering this question only requires analyzing c-fos expression in the dorsal portion of the olfactory bulb. RE: We previously quantified c-fos expression in the posterior region of the olfactory bulb (OB) (Fig.  4a,b and Supple mentary Fig. 6b). As suggested by the reviewer, we also examined the dorsal region of OB that was specifically activated by TMT. 2MT exposure induced a similar low level of c-fos expression in the dorsal region of OB in Trpa1 +/and Trpa1 -/mice (Supple me ntary Fig. 6c).
3) The authors should report their statistical analysis more clearly and include a section in the methods about it. This is particularly relevant given that in a few cases it is unclear whether the statistical tests are appropriate for the data. For example for the data in figure 4c, the authors report a one-way ANOVA for an n of 3. Not only the sample size is too small for an ANOVA, but it is unclear what is exactly the data the authors are trying to explain since they show 2 data sets for each receptor (one for TMT and another for 2MT). Is the data being pool? How? When multiple comparisons are shown (* symbols in figures) but a single value of an ANOVA is reported it is unclear which post-hoc tests were used and if in all cases correction for multiple comparisons was used. RE: As suggested by the reviewer, we include a section in the method about statistic al analysis. We are sorry for the confusion in the original Fig. 4c. In the revision, we separated this figure into 2 figures for 2MT and TMT, respectively (Fig. 5d, e). There were at le ast 3 biological replicates for each Trpa1 construct for 2MT or TMT experiments, which was normalized to wild-type construct. RE: We thank the reviewer for this excellent suggestion. Yes, we did test this, but AAV-GFP and AAV-TRPA1 injected mice showed no apparent difference in the freezing response to low dose 2MT exposure. Thus, this was probably a partial rescue because TRPA1 might have important functions in other tissues. Future studies are needed to further elucidate the specific roles of TRPA1 in the TG and other tissues with regard to predator odor-evoked innate fear/defensive responses.
5) The double staining images shown for the rescue experiment (Fig7 G and H) are not very helpful without a quantification of double-labeled cells. RE: The reviewer has an excellent point. In this AAV rescue experiment, my students encountered difficulty in performing the perfusion and double Trpa1 ISH/c-Fos IHC staining. They wasted most of the precious TG samples and ended up only with one good Trpa1/c-Fos double staining result. We now include this data in the revision (Fig. 7i), which shows high percentage of Trpa1/Fos double positive cells in AAV-TRPA1 injected TG, but not AAV-GFP injected TG. Because our remaining Trpa1 -/mice are old, it will take us 4-6 months to generate new Trpa1 -/mice and repeat this double staining experiment. We sincerely appreciate reviewer for his/her understanding.

Reviewer #2 (Remarks to the Author):
1. The first question seems partially resolved, at least in HEK cells. However, increase of c-fos expression after 2MT exposure was detected in only a fraction of TRPA1+ neurons on the TG and many of the activated TG neurons lacked Trpa1 expression (fig 6f), rising concerns about sensitivity and specificity. More evidence showing that Trpa1 neurons indeed detect TMT/2MT and their level of specificity would strengthen the conclusions. Using a more physiological model or in vivo recordings would address this point, perhaps including other Trpa1-expressing neurons such as DRG. It would also help substantially to use an experimental approach with a better temporal resolution than c-fos staining (Ca-imaging or electrophysiology recordings, for example). These experiments should clarify the specificity of TMT/2MT for Trpa1-neurons comparing to potential responses in KO controls. RE: Trigeminal ganglia (TG) contain neuron cell bodies for the 3 branches (ophthalmic, mandibular, and maxilla ry divisions) of the trigeminal nerve. TG neurons innervate the majority of craniofacial region, including the nose, mouth, and eyes. We believe that only a subset of TG neurons that innervated specific craniofacial region, such as the nasal cavity, would be responsible for 2MT sensing. Accordingly, we observed that only a specific subset of TRPA1+ neurons showed nucle ar c-Fos signals after 2MT exposure, and that only a subset of ectopic TRPA1-expressing neurons were c-Fos+ in response to 2MT in our AAV-TRPA1 TG rescued Trpa1-/-mice (Fig. 7h,i) . These observations, together with our findings, suggest that 2MT could activate a subset of TRPA1+ primary sensory neurons, which then release neuromodulators to stimulate c-fos expression in downstream TRPA1-neurons in the TG. Because TG is located at the base of the brain, it is highly challenging to perform in vivo Ca-imaging or ele ctrophysiology recordings. We think these are excellent experiments to pursue in the future, but are beyond the scope of the current paper.
2. Results using the learned fear assay (Fig 5e and suppl fig 8c,d) indicate that Trpa1-/-mice are indeed able to detect 2MT, likely via the olfactory system (Fig 5c,d). Unfortunately, no data on heterozygous or WT controls in the learned fear assay was included for comparison with the KO. This is an important control to exclude any possible reduction in olfactory performance in Trpa1-/-mice. Reduced olfaction may have an important impact on the behavior and thus, conclusions would need to be reformulated. The cookie test shown on suppl fig 8a is clearly insufficient to evaluate olfaction in Trpa1-/-. A habituation-deshabituation test using low 2MT dose (as on Fig 3) might be more appropriate in this case. In this context, showing that other odorants not detected by Trpa1 (such as those used in refs 17,47,48) can induce freezing would greatly strengthen the work. RE: We did not include WT or HET mice in 2MT learned fear experiment because they already showed very high level freezing in response to 2MT and were not further enhanced by pairing with ele ctric footshock. Besides the cookie test, Trpa1 +/and Trpa1 -/mice showed equivalent performance in the dual odor-based learned fear assay (Fig. 4d), suggesting that Trpa1 -/mice could distinguish different odors and have normal learned fear responses. As suggested by the reviewer, we performed the habituation-deshabituation test showing that Trpa1 +/and Trpa1 -/mice had a similar low detection threshold for 2MT (Fig. 4c). Taken together, these results strongly suggest that Trpa1 -/mice have a normal sense of smell. Finally, other odorants, such as non-volatile major urinary proteins (refs 47, 48), elicit only avoidance and risk assessment behaviors, but not freezing. Rather, we synthesized and tested ala rm pheromone SBT (ref 17), which is structurally related to TMT/2MT, and showed that Trpa1 -/mice were defective for SBT-evoked innate freezing behavior (Supple mentary Fig. 5d).
3. Whether Trpa1+ cells in the TG are driving the observed fear-like responses is unclear to me. First, the 2MT effect on behavior seems to be partial: background (no odor) freezing rates in WT mice ranges 0-20% (Fig 1a) whereas in 2MT-induced in Trpa1 mutants is 2-fold higher, up to 20-40% (figs 2b and 3a). This would be consistent with decreased olfaction. A no odor control in Trpa1-/-mice may clarify this point. Second, authors use a (>2s) decrease in locomotor activity as a proxy for freezing, which is assumed as a fear response. Careful observation of the movies reveals that TMT/2MT not only elicits freezing, but also sitting in distant areas from the stimulus, which can be also interpreted as avoidance. This point should be mentioned in the discussion, which now is overly oriented towards emotional fear. The "emotional" component of aversion is supposed to be lower than fear, fitting better with the classical function of TG neurons. Third, and related with the previous point, it has been reported (in ref. 13) that a subset of olfactory-activated neurons from the cortical amygdala plays a critical role in the generation of innate TMT-driven aversive behavior. Activity in this brain region in Trap1-/-mice in response to TMT/2MT should be reported to determine whether olfaction is dispensable for the display of the innate aversive-freezing behaviors. RE: We include no odor control of Trpa1 +/and Trpa1 -/mice, both of which showed 0-20% baseline freezing rate (Supple mentary Fig. 5c). By contrast, Trpa1 -/mice showed 20-30% freezing rate as compare to 55-90% freezing rate of WT or Trpa1 +/littermates in response to 2MT exposure. The results suggest that TRPA1 plays a crucial role in 2MT-evoked freezing behavior. The residual freezing response of Trpa1 -/mice toward 2MT is probably attributed to a functional olfactory system. Moreover, we showed that Trpa1 -/mice were also defective for the avoidance, flight, risk assessment behaviors in response to low dose 2MT (Fig. 3d,e) and snake skin molt (Fig. 3f,g). As suggested by reviewer, we include in the revision more discussion of these innate defensive behaviors in addition to freezing. Finally, we showed that 2MT evoked a similar low level of c-fos expression in the cortical amygdala (CA) of Trpa1 +/and Trpa1 -/mice (Fig. 4a,b). Importantly, habituation-deshabituation test showed a similar low detection threshold for 2MT in Trpa1 +/and Trpa1 -/mice. These results strongly suggest that Trpa1 -/mice have a normal sense of smell. It should also be emphasized that we believe both the trigeminal and olfactory systems play dual roles in innate aversive-freezing behaviors.
Minor points: 1. In fig 4 and suppl fig 6, can responses to TMT/2MT be eliminated using a specific inhibitor for Trpa1 such as HC-030031? RE: We thank reviewer for this excellent suggestion. We showed that inhibition of TRPA1 by HC-030031 blocked 2MT-evoked Ca 2+ transients in transfected HEK293 cells (Supple mentary Fig. 7b). pedigree (males and females) that carried 40-60 mutations/exome, including Trpv1 mutation. These ENU mutant mic e showed more variable phenotypes because of two major reasons: 1) female mice generally showed less freezing response than male mice upon 2MT exposure; 2) ENU mutant mic e previously endured nine different genetic screens, including a brutal chemical-induced colitis screen, which could increase the individual variations in their behaviors in our innate fear assay.