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. 2018 May 23;9(1):2041.
doi: 10.1038/s41467-018-04324-3.

Large-scale Forward Genetics Screening Identifies Trpa1 as a Chemosensor for Predator Odor-Evoked Innate Fear Behaviors

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Free PMC article

Large-scale Forward Genetics Screening Identifies Trpa1 as a Chemosensor for Predator Odor-Evoked Innate Fear Behaviors

Yibing Wang et al. Nat Commun. .
Free PMC article

Abstract

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Development of a highly robust innate fear assay. a A schematic of 2MT-evoked innate fear assay (left). A quantitative graph showing the distribution of freezing rates of C57BL/6J males with or without (20 µl = 2.1 × 104 mole) 2MT exposure (right). b A graph showing the distribution of freezing rates (average 20 min) in C57BL/6J males (>15 week old) at different doses of 2MT exposure. c A graph showing the distribution of freezing rates (average 20 min) at different ages of C57BL/6J males exposed to 20 µl of 2MT. d A graph showing the distribution of freezing rates for different assay duration using C57BL/6J males. −3SD, 3 standard deviation (SD) below the normal mean of freezing rates upon 2MT exposure. The numbers in parentheses indicate the sample numbers for individual conditions
Fig. 2
Fig. 2
Identification of fearless (Trpa1) mutant mice by forward genetics screening. a A flowchart of the recessive fear screen. b A graph of the fearless mutant pedigree consisting of seven reference (REF), six heterozygous (HET), and four phenovariant (VAR) individuals. A separate group of wild-type (WT) mice were included as controls every week. c A Manhattan plot showing a strong linkage (P = 2.13 × 1011) between the Trpa1 mutation and fearless phenotype. Horizontal yellow and blue lines represent the thresholds of P < 0.05 without or with Bonferroni correction, respectively. d The causative mutation was identified by sequencing the Trpa1 gene of REF, HET, and VAR mice. e A schematic of partial exon/intron structure of Trpa1 gene. The 5′ splice site (GT to GC) mutation results in the skipping of exon 15 and introduction of a premature stop codon. f, g Semi-quantitative (f) and quantitative (g) RT-PCR analysis showing that fearless mutant Trpa1 mRNA is diminished possibly owing to nonsense-mediated decay. Data are presented as mean ± SEM (n = 4, Student’s t-test, ***P < 0.001). h A schematic of the domain structure of Trpa1 protein, with the stop sign marking the premature stop codon and dash line representing the domains missing in mutant Trpa1 protein
Fig. 3
Fig. 3
Trpa1 mediates 2MT/TMT/snake skin-evoked innate fear/defensive responses. a, b Wild-type, heterozygous, and homozygous Trpa1 (a) and Trpv1 (b) knockout mice were examined for innate freezing behaviors evoked by 2MT or TMT. Data are presented as mean ± SEM (Student’s t-test, ***P < 0.001; **P < 0.01; ns not significant). c Plasma concentration of stress hormone corticosterone was measured in Trpa1+/− and Trpa1−/− mice following exposure to H2O or 2MT for 20 min. Data are presented as mean ± SEM (n = 6, two-way ANOVA test, **P < 0.01). d Representative avoidance tracks of Trpa1+/− and Trpa1−/− mice in response to low concentration (1.05 × 106 mole) of 2MT exposure in the test cage. The black box indicates the position of 2MT-containing filter paper. e Quantitative analysis of freezing, risk assessment, flight, and investigation behaviors of Trpa1+/− and Trpa1−/− mice in testing cages in response to low-dose 2MT exposure. Data are presented as mean ± SEM (n = 7, Student’s t-test, ***P < 0.001; **P < 0.01, *P < 0.05). f An image depicting the snake skin-evoked innate fear assay in the home cage. g Quantitative analysis of snake skin-evoked freezing, risk assessment, avoidance, flight, and investigation behaviors of Trpa1+/− and Trpa1−/− mice. Data are presented as mean ± SEM (n = 6, Student’s t-test, ***P < 0.001; **P < 0.01)
Fig. 4
Fig. 4
Trpa1−/− mice can smell and learn to fear 2MT. a Representative images showing c-fos mRNA in situ hybridization (ISH) of the central nucleus of amygdala (CeA), paraventricular nucleus (PVN) of hypothalamus, ventral periaqueductal gray (vPAG), olfactory bulb (OB), and cortical amygdala (CoA) regions of the brains of 2MT-exposed Trpa1+/− and Trpa1−/− mice (bar: 100 µm). b Quantitative analysis of c-fos-positive neurons in the CeA, PVN, vPAG, OB, and CoA of Trpa1+/− and Trpa1−/− mouse brains (a). The relative measure (%) of c-fos signals of Trpa1−/− samples is normalized to those of Trpa1+/− controls. Data are presented as mean ± SEM (n = 4, Student’s t-test, ***P < 0.001; ns not significant). c Habituation-dishabituation test results of Trpa1+/− and Trpa1−/− mice exposed to filter papers containing H2O (trials 1–4) or 2MT (trial 5). 2MT dose was shown above the left and right graphs (left graph: n = 6 for both Trpa1+/− and Trpa1−/− mice, Student’s t-test; ns not significant; right graph: n = 5 for Trpa1+/− mice and n = 4 for Trpa1−/− mice, **P < 0.01). d Dual odor-based learned fear assay was performed with Trpa1+/frl and Trpa1frl/frl mutant mice by pairing anisole with electric footshock and using eugenol as a negative control. Data are presented as mean ± SEM (n = 10, Student’s t-test, ***P < 0.001; ns not significant). e Trpa1−/− mice were untrained (−FS) or trained (+FS) to fear 2MT by pairing 2MT exposure with electric footshocks. Eugenol was used as a control odor. Data are presented as mean ± SEM (n = 6, Student’s t-test, ***P < 0.001; **P < 0.01; *P < 0.05)
Fig. 5
Fig. 5
Trpa1 acts as a chemosensor for 2MT/TMT. a Twenty-four hours after transfection of the Trpa1-P2A-mCherry construct, Ca2+ imaging was performed in HEK293T cells upon exposure to 1, 10, 100, 1000 μM of 2MO, 2MT, or TMT. b, c 2MT (100 μM, b) or TMT (50 μM, c)-evoked Ca2+ response curves of HEK293T cells after co-transfection of the mCherry plasmid with each of 12 constructs expressing different mammalian TRP channels (h human, r rat, m mouse). d, e Quantitative analysis of wild-type (WT) and mutant Trpa1 activities in response to 2MT (100 μM, d) or TMT (50 μM, e) by Ca2+ imaging in transfected HEK293T cells. Y-axis, F/F0 ratio normalized to WT construct. Data are presented as mean ± SEM (n = 3 biological replicates). Pairwise comparisons were made for each mutant Trpa1 to wild-type Trpa1 using two-way unpaired Student’s t-test. Multiple comparisons were controlled by Bonferroni correction with unadjusted P-value cutoff being 0.005 (***), 0.01 (**), and 0.05 (*). f A schematic of in vivo labeling of transfected HA-Trpa1 with alkynyl-TMT followed by biotin conjugation via click chemistry and streptavidin pull down. g, h Immunoblots showing that alkynyl-TMT (A-TMT), but not TMT, could pull down HA-Trpa1 (g), and that alkynyl-TMT could pull down C415S mutant less efficiently than wild-type HA-Trpa1 (h) from transfected HEK293T cells
Fig. 6
Fig. 6
Trpa1 is essential for 2MT sensing by a subset of TG neurons. a, b Immunohistochemistry (IHC) (a) and quantitation (b) of c-Fos-expressing TG neurons of Trpa1+/frl and Trpa1frl/frl mice after H2O or 2MT exposure. Data are presented as mean ± SEM (n = 4, Student’s t-test, *P < 0.05). c, d In situ hybridization (ISH) (a) and quantitation (b) of c-fos-expressing neurons in the Sp5C of Trpa1+/− and Trpa1−/− mice after 2MT exposure. The relative measure (%) of c-fos signals of Trpa1−/− samples is normalized to those of Trpa1+/− samples (d). Data are presented as mean ± SEM (n = 4, Student’s t-test, ***P < 0.001). e, f Double staining (e) and quantitation (f) of Trpa1+, c-Fos+ double positive TG neurons of Trpa1+/frl mice after 2MT exposure. Scale bars are 100 µm in a and 10 µm in c and e
Fig. 7
Fig. 7
Trpa1+ TG neurons contribute critically to 2MT-evoked innate freezing. a Hematoxylin and eosin staining of the control and lesioned TG of wild-type mouse after unilateral lesion (ulTGx). b Representative images showing c-fos ISH signals in the Sp5C regions of ulTGx mice after 2MT exposure. Enlarged images of the control (white arrow) and lesion sides (black arrow) are shown in the lower panels. Scale bars are 100 µm in a and b. c Quantitative analysis of c-fos ISH signals in the Sp5C regions of the control and lesion sides of ulTGx mice after 2MT exposure. The relative measure (%) of c-fos signals of the lesion side is normalized to that of control side. Data are presented as mean ± SEM (n = 8, Student’s t-test, **P< 0.01). d Quantitative analysis of 2MT-evoked freezing behavior in the sham (black) and ulTGx (red) mice. Data are presented as mean ± SEM (n = 8, Student’s t-test, **P < 0.01, ***P < 0.001). e Quantification of average freezing rate in the sham (black) and ulTGx (red) mice during 10 min of 2MT treatment. Data are presented as mean ± SEM (n = 8, Student’s t-test, *P< 0.05). f A schematic of bilateral injection of AAV-GFP or AAV-Trpa1 into the TG of Trpa1−/− mice. g Representative IHC images of AAV-GFP-infected TG sections of Trpa1−/− mice. GFP (green); c-Fos (red); DAPI (blue). h Representative images of double Trpa1/c-Fos staining of AAV-Trpa1-infected TG sections of Trpa1−/− mice. Trpa1 ISH (purple); c-Fos IHC (brown). Arrowheads indicate Trpa1+/c-Fos+ TG neurons. Insets are high magnification images. Scale bar is 20 µm. i Quantitation of 2MT-evoked GFP+, c-Fos+, or Trpa1+/c-Fos+ TG double positive neurons in AAV-GFP or AAV-Trpa1-infected TG, respectively. j, k Quantitative analysis of 2MT-evoked freezing behavior in the AAV-GFP (blue) and AAV-Trpa1 (red)-injected mice. Data are presented as mean ± SEM (n = 6, Student’s t-test, *P < 0.05; **P < 0.01)

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