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. 2020 May 29;6(22):eaaz6868.
doi: 10.1126/sciadv.aaz6868. eCollection 2020 May.

Physiology-forward identification of bile acid-sensitive vomeronasal receptors

Wen Mai Wong  1 , Jie Cao  1 , Xingjian Zhang  1 , Wayne I Doyle  1 , Luis L Mercado  2 , Laurent Gautron  2 , Julian P Meeks  1
Affiliations

Physiology-forward identification of bile acid-sensitive vomeronasal receptors

Wen Mai Wong et al. Sci Adv. .

Abstract

The mouse accessory olfactory system (AOS) supports social and reproductive behavior through the sensation of environmental chemosignals. A growing number of excreted steroids have been shown to be potent AOS cues, including bile acids (BAs) found in feces. As is still the case with most AOS ligands, the specific receptors used by vomeronasal sensory neurons (VSNs) to detect BAs remain unknown. To identify VSN BA receptors, we first performed a deep analysis of VSN BA tuning using volumetric GCaMP6f/s Ca2+ imaging. These experiments revealed multiple populations of BA-receptive VSNs with submicromolar sensitivities. We then developed a new physiology-forward approach for identifying AOS ligand-receptor interactions, which we call Fluorescence Live Imaging for Cell Capture and RNA sequencing, or FLICCR-seq. FLICCR-seq analysis revealed five specific V1R family receptors enriched in BA-sensitive VSNs. These studies introduce a powerful new approach for ligand-receptor matching and reveal biological mechanisms underlying mammalian BA chemosensation.

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Figures

Fig. 1
Fig. 1. Evaluating VSN responses to monomolecular BAs with population VSN Ca2+ imaging via OCPI microscopy.
(A) OCPI (light sheet) microscopy imaging setup. Thousands of VSNs in the intact vomeronasal epithelium were imaged via this setup to measure the responses across a panel of ligands. (B) Experimental stimulus panel consisting of five test concentrations of two BAs (CA, cholic acid; DCA, deoxycholic acid) along with positive controls (FF, dilute female mouse feces) and negative controls (Ringer’s). (C) Representative fluorescence intensity traces of volumetric regions of interest (ROIs) isolating two individual VSNs over the course of one experiment. Colored bars represent ligand presentation corresponding to ligands depicted in (B). Each stimulus presentation lasted five stacks (~15 s), with 15 stacks (~45 s) between stimulus presentations. (D) Representative across-trial responses of the same VSNs in (C). Bolded traces show across-trial mean waveforms, with red line color indicating a statistically significant increase compared to Ringer’s control trials.
Fig. 2
Fig. 2. VSN responses to CA and DCA.
(A and B) Representative colorized images of VSNs response (ΔF/F) in a single frame of and OCPI image stack (left). The responses to 10 μM CA (red), 1 μM CA (green), and 0.1 μM CA (blue) are shown in (A). The responses of VSNs to 10 μM CA (red), 1 μM CA (green), and 10 μM DCA (blue) are shown in (B). Across-trial VSN responses are plotted as individual traces. Bolded trace indicates the mean response across all stimulus repeats. Responses in the cyan box correspond to the VSN indicated by the cyan arrow, those in the orange box correspond to those indicated by orange arrows, and traces in the magenta box correspond to the neuron indicated by the magenta arrow. (C) Clustered heat map of VSN response to CA and DCA at varying concentrations. Each column indicates an individual neuronal response. Cluster divisions are indicated by black vertical lines. Arrows above the heat map highlight the clusters in which neurons from (A) and (B) fall. Shown are the responses of 1222 VSNs from three OMPxAi96 animals. All experiments included at least three randomized, interleaved stimulus repeats. (D) Chemical structure diagrams of CA and DCA. Red annotations indicate key differences. (E) Heat map of cluster separation. The map indicates the pairwise discriminability index (d′) between each cluster [based on linear discriminant analysis (LDA)]. White hue indicates the P = 0.05 cutoff, and shades of red indicate higher discriminability (lower P values). For all these pairwise comparisons, P < 0.05.
Fig. 3
Fig. 3. VSN responses to CA and LCA.
(A and B) Representative colorized images of VSNs (ΔF/F) in a single frame of an OCPI image stack (left). The responses to 10 μM CA (red), 1 μM CA (green), and 10 μM LCA (blue) are shown in (A). The responses of VSNs to 10 μM CA (red), 1 μM CA (green), and 0.1 μM CA (blue) are shown in (B). Across-trial VSN responses are plotted as individual traces. Bolded trace indicates the mean response across all stimulus repeats. Responses in the cyan box correspond to the VSN indicated by the cyan arrow, while those in the orange box correspond to those indicated by orange arrows. (C) Clustered heat map of VSN response to CA and LCA at varying concentrations. Each column indicates an individual neuronal response. Cluster divisions are indicated by black vertical lines. Arrows above the heat map highlight the clusters in which neurons from (A) and (B) fall. Shown are the responses of 1362 VSNs from three OMPxAi96 animals. All experiments included at least three randomized, interleaved stimulus repeats. (D) Chemical structure diagrams of CA and LCA. Red annotations indicate key differences. (E) Heat map of cluster separation. The map indicates the pairwise discriminability index (d′) between each cluster (based on LDA). White hue indicates the P = 0.05 cutoff, and shades of red indicate higher discriminability (lower P values). For all these pairwise comparisons, P < 0.05.
Fig. 4
Fig. 4. VSN responses to a panel of monomolecular ligands and sulfated steroids.
(A and B) Representative colorized images of VSN responses (ΔF/F) in a single frame of an OCPI image stack (left). The responses to 10 μM CA (red), 10 μM DCA (green), and 10 μM Q1570 (blue) are shown in (A). The responses of VSNs to 10 μM CDCA (red), 10 μM A6940 (green), and 10 μM Q1570 (blue) are shown in (B). Across-trial VSN responses are plotted as individual traces. Bolded trace indicates the mean response across all stimulus repeats. Responses in the cyan box correspond to the VSN indicated by the cyan arrow, those in the orange box correspond to those indicated by orange arrows, and traces in the magenta box correspond to the neuron indicated by the magenta arrow. (C) Clustered heat map of VSN response to a panel of monomolecular ligands. Each ligand is shown at left to highlight structural differences, with red annotations indicating key differences among BAs. Each column indicates an individual neuronal response. Cluster divisions are indicated by black vertical lines. Arrows above the heat map highlight the clusters in which neurons from (A) and (B) fall. Shown are the responses of 890 VSNs from three OMPxAi96 animals. All experiments included at least three randomized, interleaved stimulus repeats. (D) Chemical structure diagrams of all ligands used in this experiment. Red annotations indicate key differences among BAs. (E) Heat map of cluster separation. The map indicates the pairwise discriminability index (d′) between each cluster (based on LDA). White hue indicates the P = 0.05 cutoff, and shades of red indicate higher discriminability (lower P values). For all these pairwise comparisons, P < 0.05.
Fig. 5
Fig. 5. Function-forward selection of BA-sensitive VSNs for scRNAseq.
(A) Overview of FLICCR-seq experimental setup. VNO slices from OMP-GCaMP6s mice were stimulated with 1 μM BAs a minimum of three times. BA-responsive cells are plucked with a modified glass patch pipette and processed for scRNAseq. (B) Representative fluorescence images of 1 μM CA-responsive VSNs. The VSNs marked with ‡ and † were selected and processed for scRNAseq. DIC, differential interference contrast. (C) Representative images of VSN selection during active stimulation (in this trial with 1 μM DCA). (D) UMAP multidimensional scaling of 158 samples, including 145 VSNs collected using these methods. Clusters 0 to 2 represent subsets of a larger group, whereas cluster 3 identifies a smaller, isolated cluster. Cluster 4 (gray, asterisk) contained RNA kit control samples and are accordingly distinct from VSNs. (E) Left: UMAP multidimensional scaling with each cell colorized based on expression of Gnai2 (Gαi, magenta) and Gnao1 (Gαo, cyan), genes that are associated with V1R- and V2R-expressing VRs, respectively. This analysis indicates that clusters 0 to 2 contain V1R-expressing VRs, while cluster 3 contains mostly V2R-expressing VRs. Right: Same UMAP scaling with each cell colorized based on the expression of Ano1 (Anoctamin 1, also known as TMEM16α) and Trpc2 (cyan), ion channels that have been studied in VSNs. Expression of Trpc2 or both Trpc2 and Ano1 was detected in almost all cells. (F) Genome viewer visualization of 18 representative VSNs showing the mapped scRNAseq reads for Trpc2, selected V1Rs and V2Rs, and G protein α subunits. The rows marked with ‡ and † reflect the cells indicated in (B) and (C). Note that the number of reads for individual VRs in each VSN was often stronger than for reference genes (e.g., Trpc2, Gapdh, and Actb).
Fig. 6
Fig. 6. Identification of BA-sensitive VRs.
(A) Clustered heat map showing the gene expression for all 158 analyzed samples. Rows are individual transcripts, with divisions between functional subgroups indicated by a horizontal dotted line. At the top are control genes (see Materials and Methods). At the bottom is a lookup table of responsivity. Cyan indicates that the sample contained a VSN that was reliably responsive to the stimulus, dark blue indicates an inconsistent response, black indicates no response, and gray indicates that the ligand was not tested on that VSN. For cell amount (“Cell amt.”), green indicates a single cell was cleanly picked, orange indicates that additional cell material was visible in/on the pipette tip, red indicates a bulk tissue extraction, and black indicates RNA kit control samples. (B) Heat map plot of the top four differentially expressed genes for BA-responsive VSNs. In each subplot, the colored horizontal bars indicate the responsivity, with colors representing the same qualities as in (A). VSNs with unclear responsivity for the specified ligand were excluded from these plots. Expression levels (log counts/10,000) were normalized by the maximum values per gene across all samples. (C) Duplex RNA in situ hybridization micrographs for combined immediate-early gene (IEG) probes (Egr1 and Fos; blue stain) and test VRs (red stain). Mice were exposed in vivo to control saline or a mixture of BAs at 0.1 or 1 mM to stimulate VSNs. (D) Violin plot of the density of VR staining across VNO sections from 30 mice (15 males and 15 females). (E) Violin plot of the density of IEG staining (24 mice, three males and three females per condition, and four conditions). (F) Violin plots of the normalized number of colocalized IEG+ puncta per VR+ cell following in vivo exposure to BAs or female mouse fecal extracts. Multiple VNO sections were analyzed from 24 mice total [same cohort as in (E)]. †P < 0.1 and *P < 0.05 (Kruskal-Wallis test followed by multiple comparisons test). n.s., not significant.
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