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Featured ArticleArticles, Cellular/Molecular

In Vivo Identification of Eugenol-Responsive and Muscone-Responsive Mouse Odorant Receptors

Timothy S. McClintock, Kaylin Adipietro, William B. Titlow, Patrick Breheny, Andreas Walz, Peter Mombaerts and Hiroaki Matsunami
Journal of Neuroscience 19 November 2014, 34 (47) 15669-15678; https://doi.org/10.1523/JNEUROSCI.3625-14.2014
Timothy S. McClintock
1Department of Physiology, University of Kentucky, Lexington, Kentucky 40536,
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  • ORCID record for Timothy S. McClintock
Kaylin Adipietro
5Department of Molecular Genetics and Microbiology and
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William B. Titlow
1Department of Physiology, University of Kentucky, Lexington, Kentucky 40536,
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Patrick Breheny
2Department of Biostatistics, University of Iowa, Iowa City, Iowa 52242,
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Andreas Walz
3Rockefeller University, New York, New York 10065,
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Peter Mombaerts
3Rockefeller University, New York, New York 10065,
4Max Planck Research Unit for Neurogenetics, D-60438 Frankfurt, Germany,
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Hiroaki Matsunami
5Department of Molecular Genetics and Microbiology and
6Duke Institute for Brain Sciences, Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710
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  • Figure 1.
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    Figure 1.

    The gene-targeted S100a5–tauGFP mouse strain allows for activity-dependent fluorescent marking of OSNs. A, The design of the targeted mutation of the mouse S100a5 locus, replacing the coding exons with tauGFP. Boxes, Exons; light blue, coding sequence; dark blue, noncoding sequence; triangles, loxP sites; arrow, translation start site. B, Exposure for 4.5 s detects fluorescent OSNs in a 12 μm coronal section at midseptum, showing the mosaic pattern and variable levels of GFP expression in a male mouse, 25 d postnatal, that is wild type for the Cnga2 locus. C, Exposure for 49 s reveals a near absence of fluorescent OSNs in a Cnga2-deficient male mouse, 25 d postnatal. Scale bars, 100 μm. D, FACS of dissociated cells from olfactory mucosae of a wild-type C57BL/6 mouse is used to set sorting gates (circumscribed with magenta lines) for subsequent capture of GFP+ and GFP− cells from S100a5–tauGFP mice. E, F, FACS capture of GFP+ and GFP− cells from a set of four S100a5–tauGFP mice exposed to vehicle (E) and four mice exposed in parallel to muscone (F). G, In the in vivo assay, OSNs that exhibit GFP fluorescence above background are captured in the GFP+ sample. The distribution of each OSN subtype in GFP+ and GFP− FACS samples from S100a5–tauGFP mice exposed to an odorant ligand differs from that of S100a5–tauGFP mice exposed to vehicle only in those few OSN subtypes that express ORs responsive to the odorant ligand, as exemplified by OR6 in this diagram. Odorant-evoked activity in OR6-expressing OSNs results in an increase of the fraction of these OSNs with GFP fluorescence above background, thus causing a redistribution of these OSNs from the GFP− FACS sample into the GFP+ FACS sample. Differing intensities of GFP fluorescence above background exist among OSNs but are not relevant in this assay, which is based on a binary grouping of fluorescence intensities (GFP− vs GFP+).

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    Figure 2.

    Eugenol exposure has significant effects on three ORs in S100a5–tauGFP mice. A, The distribution of OR mRNAs between GFP+ and GFP− samples is consistent across experiments. GFP+/GFP− ratios from mice exposed to vehicle (mineral oil) from four replications, distinguished by color (red, blue, green, and black), plotted against the mean GFP+/GFP− ratios of these four vehicle treatments. B, Mean OR mRNA GFP+/GFP− ratio values after exposure to eugenol plotted against the mean ratios after exposure to vehicle (n = 4 groups of mice). Three ORs (Olfr961, Olfr958, and Olfr960) show significant elevation (FDR < 0.10) after eugenol exposure. To illustrate the change, their GFP+/GFP− ratio values in this experiment (green diamonds) are compared with the GFP+/GFP− ratio coordinates in other experiments that did not use eugenol (blue circles). C, Chemical structure of eugenol. D, Mean GFP+/GFP− ratios and corresponding FDR probabilities for the seven ORs with FDR probabilities <0.5.

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    Figure 3.

    Heterologous expression assay data for ORs responsive to eugenol. A–C, Eugenol concentration–response relationships for Olfr961, Olfr958, and Olfr960, previously identified eugenol-responsive ORs that gave significant differences in the in vivo assay. D, Eugenol concentration–response relationship for Olfr73, the first eugenol-responsive OR to be identified (Kajiya et al., 2001).

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    Figure 4.

    Muscone exposure has significant effects on five ORs in S100a5–tauGFP mice. A, Chemical structure of muscone. B, Mean OR mRNA GFP+/GFP− ratios after exposure to muscone plotted against the mean ratios after exposure to vehicle (n = 4 groups of mice). Five ORs (Olfr1440, Olfr1433, Olfr235, Olfr1431, and Olfr1437) show significant differences (FDR < 0.10) after muscone exposure (green triangles) compared with vehicle exposure. Blue circles reflect the GFP+/GFP− ratio coordinates of these five ORs in other experiments that did not use muscone. C, Mean GFP+/GFP− ratios and corresponding FDR probabilities for the 13 ORs with FDR probabilities <0.5. D, A portion of the mouse OR phylogenetic tree depicting the relatedness of the members of the MOR214 and MOR215 mouse OR families that gave significant responses to muscone (green).

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    Figure 5.

    Confirmation of muscone responses from two mouse ORs by heterologous expression. A, Olfr1440. B, Olfr235. C, Rho-pCI, empty plasmid vector control. IPM used as vehicle.

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    Figure 6.

    Activation of muscone-responsive mouse and human ORs by certain macrocyclic musk odorants in heterologous expression assays. A, OR5AN1, a human OR, responded to the macrocyclic ketone musks muscone and Exaltone and to the macrocyclic lactone musk Exaltolide. B, The OR5AN1 response to muscone is stronger than responses of mouse ORs. C, Olfr1440 and Olfr235 responses to muscone. D, E, Polycyclic musk odors fail to evoke responses different from the plasmid vector negative control, Rho-pCI, in cells transfected with OR5AN1 or the mouse ORs responsive to muscone. Data for some ORs cannot be seen because of overlap of the curves. F, Astrotone, a macrocyclic diester musk odorant, also fails to evoke responses different from the plasmid vector negative control, Rho-pCI, in cells transfected with OR5AN1 or the mouse ORs responsive to muscone. Data for some ORs cannot be seen because of overlap of the curves.

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    Figure 7.

    Distribution of the 50 lowest FDR probabilities from the in vivo eugenol and muscone experiments illustrate the ability of the assay to separate odorant-responsive receptors from nonresponders. Receptors with FDR probabilities <0.001 were truncated at 0.001.

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    Figure 8.

    Heterologous expression assay data for additional ORs responsive to eugenol or muscone. A–E, Eugenol concentration–response relationships for five eugenol-responsive ORs that were previously orphan receptors. F, G, Muscone concentration–response relationships for two muscone-responsive ORs that were previously orphan receptors. Rho–pCI, Expression plasmid used as a negative control. IPM used as vehicle.

Tables

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    Table 1.

    ORs tested in the Hana3A cell heterologous expression assay

    EugenolMuscone
    OlfrMORFDROlfrMORFDR
    Olfr961*MOR224-50.0002Olfr1433MOR214-40.0000
    Olfr958*MOR224-90.0188Olfr1440*MOR215-10.0000
    Olfr960*MOR188-50.0728Olfr235*MOR214-30.0000
    Olfr1101MOR179-30.3015Olfr1437MOR214-60.0024
    Olfr1085MOR191-10.3283Olfr1431MOR214-50.0074
    Olfr1501MOR212-30.3516Olfr1020MOR201-20.4447
    Olfr97MOR156-20.4372Olfr769MOR114-40.4480
    Olfr1160MOR173-10.9179Olfr867MOR143-20.4587
    Olfr610*MOR9-20.9197Olfr250MOR170-140.4630
    Olfr1052MOR172-10.9245Olfr1257MOR232-10.4773
    Olfr1234*MOR231-20.9832Olfr1496MOR127-10.4837
    Olfr73*MOR174-91.0000Olfr74*MOR174-40.5204
    Olfr151MOR171-21.0000Olfr488MOR204-150.5354
    Olfr1031MOR200-11.0000Olfr816*MOR113-10.6397
    Olfr178*MOR184-61.0000Olfr394MOR135-80.8547
    Olfr922MOR161-31.0000Olfr1213MOR233-71.0000
    Olfr1274MOR228-41.0000
    Olfr821MOR109-11.0000
    Olfr811MOR110-61.0000
    Olfr1090MOR188-41.0000
    Olfr1325MOR102-11.0000
    Olfr1496MOR127-11.0000
    Olfr904MOR167-31.0000
    Olfr591MOR24-11.0000
    Olfr215MOR119-21.0000
    Olfr432*MOR123-21.0000
    Olfr1054MOR188-21.0000
    Olfr1014MOR213-51.0000
    Olfr360MOR159-11.0000
    Olfr913*MOR165-101.0000
    • ↵*Positive responses.

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The Journal of Neuroscience: 34 (47)
Journal of Neuroscience
Vol. 34, Issue 47
19 Nov 2014
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In Vivo Identification of Eugenol-Responsive and Muscone-Responsive Mouse Odorant Receptors
Timothy S. McClintock, Kaylin Adipietro, William B. Titlow, Patrick Breheny, Andreas Walz, Peter Mombaerts, Hiroaki Matsunami
Journal of Neuroscience 19 November 2014, 34 (47) 15669-15678; DOI: 10.1523/JNEUROSCI.3625-14.2014

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In Vivo Identification of Eugenol-Responsive and Muscone-Responsive Mouse Odorant Receptors
Timothy S. McClintock, Kaylin Adipietro, William B. Titlow, Patrick Breheny, Andreas Walz, Peter Mombaerts, Hiroaki Matsunami
Journal of Neuroscience 19 November 2014, 34 (47) 15669-15678; DOI: 10.1523/JNEUROSCI.3625-14.2014
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Keywords

  • cell sorting
  • expression profiling
  • G-protein coupled receptor
  • odor detection
  • olfaction
  • sensory coding

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