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, 2 (1), 76-88

An Olfactory Subsystem That Mediates High-Sensitivity Detection of Volatile Amines

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An Olfactory Subsystem That Mediates High-Sensitivity Detection of Volatile Amines

Rodrigo Pacifico et al. Cell Rep.

Abstract

Olfactory stimuli are detected by over 1,000 odorant receptors in mice, with each receptor being mapped to specific glomeruli in the olfactory bulb. The trace amine-associated receptors (TAARs) are a small family of evolutionarily conserved olfactory receptors whose contribution to olfaction remains enigmatic. Here, we show that a majority of the TAARs are mapped to a discrete subset of glomeruli in the dorsal olfactory bulb of the mouse. This TAAR projection is distinct from the previously described class I and class II domains, and is formed by a sensory neuron population that is restricted to express TAAR genes prior to choice. We also show that the dorsal TAAR glomeruli are selectively activated by amines at low concentrations. Our data uncover a hard-wired, parallel input stream in the main olfactory pathway that is specialized for the detection of volatile amines.

Figures

Figure 1
Figure 1. Gene targeting reveals TAAR projections to the olfactory bulb
(A) (Top) Diagram of the TAAR gene cluster. Polygons indicate gene orientation; olfactory TAARs are shown in black, non-olfactory Taar1 in white. Expression in the dorsal or ventral epithelium is indicated. (Bottom) Structure of Taar3 and Taar4 loci showing coding sequences (white boxes), non-translated regions (black boxes) and transcription start sites (arrows). Targeted insertions contain an internal ribosome entry site (grey box marked, “i”) and coding sequences for fluorescent markers (colored boxes). Black triangles indicate the location and orientation of loxP sites. (B1) Medial view of olfactory epithelium in a T4-RFP/T4-ChYFP mouse. (B2) Higher magnification shows that OSNs are either green or red. (B3) Detailed view of an epithelial section showing two neighboring OSNs, each expressing one of the two Taar4 alleles. (C) Dorsal view of the olfactory bulbs of a heterozygous T4-RFP mouse showing axonal projections and glomeruli formed by tagged OSNs. (D) Dorsal view of the bulbs of a heterozygous T4-ChYFP mouse. (E) High magnification image of a lateral glomerulus in a compound heterozygous T4-RFP/T4-ChYFP mouse. Axons coalesce into the same glomerulus. (F) Dorsal view of the bulbs of a heterozygous T3-YFP mouse. (G) Dorsal view of the bulbs of a double heterozygous T3-YFP/T4-RFP mouse. Boxed area is shown in (H). (H) Higher magnification image of lateral glomeruli in T3-YFP/T4-RFP mice. (I) Right olfactory bulbs from three T4-RFP mice illustrating different patterns of glomerular convergence by T4 axons. Arrowhead points to a “fused” glomerulus (receiving both lateral and medial innervation). Anterior and medial are indicated. (J) Positions of medial (red circles), lateral (blue circles), and “fused” glomeruli (green squares) taken from the left and right olfactory bulb across animals (n=62 bulbs, ages P15 to P75). Glomerular positions were measured with respect to the length and width of the bulbs and projected onto a bulb of average size. Scale bar = 200 μm in B1, 65 μm in B2, 20 μm in B3, 350 μm in C, D, F and G, 90 μm in E, 80 μm in H, and 250 μm in I. See also Figures S1 and S2.
Figure 2
Figure 2. T4 glomeruli lie outside DII and are closely associated with DI
(A) Medial view of the olfactory epithelium showing overlapping distribution of ΔS50 (class I) and ΔM72 (class II) OSNs in the dorsal epithelium. (B) Dorsal view of the olfactory bulbs in which ΔS50 and ΔM72 axons innervate DI (yellow) and DII (red). (C) Dorsal view of the bulbs of a P-LacZ Tg+; T4-ChYFP heterozygous mouse showing DII axons (red) and T4 glomeruli (green). T4 glomeruli fall outside of DII. (D) Coronal section through the bulb of a P-LacZ Tg+; T4-ChYFP heterozygous mouse showing a T4 glomerulus (green) in the LacZ-negative region. The boundary between this region and DII (red) is indicated by arrowheads. Nuclei are stained with TOPRO-3 (blue). (E) Dorsal view of the bulbs of a ΔS50-YFP; T4-RFP double heterozygous mouse showing the DI axons (yellow) and T4 glomeruli (red). (F) Coronal section showing absence of innervation of T4 glomerulus (red) by ΔS50-YFP axons (yellow). Counterstained with TOPRO-3 (blue). Scale bar = 260 μm in A; 500 μm in B, C, E; 130 μm in D; 30 μm in F.
Figure 3
Figure 3. TAAR coding sequence deletions reveal a third domain
(A) Gene targeting to generate three TAAR-deletion alleles. Non-coding regions are shown as black boxes. Transcription start sites shown as arrows. The coding sequence (white box) of Taar4 is replaced with either Venus YFP (yellow) or gap-Cherry (red); Taar9 is replaced with Cerulean CFP. LoxP sites are shown as black triangles. (B) Dorsal view of the olfactory bulbs of a heterozygous ΔT4-YFP mouse showing a cluster of glomeruli (green) located in the caudal bulb. (C) Dorsal view of the bulbs of a homozygous ΔT9-CFP mouse. (D) Dorsal view of the bulbs of a P-LacZ Tg+; ΔT4-YFP heterozygous mouse. The axons and glomeruli labeled by ΔT4-YFP axons (green) are located outside of the DII domain (red). (E)Dorsal view of the bulbs of a ΔS50-YFP; ΔT4-RFP double heterozygous mouse showing that the glomeruli labeled by the ΔT4-RFPallele (red) are distinct from DI (yellow). (F) Dorsal view of the bulbs of a P-LacZ Tg+; ΔS50-YFP; ΔT9-CFPtriple-mutant mouse showing that the region labeled by the T9-CFPallele (cyan) is largely separate from DI (yellow) and DII (red). (G) Coronal section through the bulb of a P-LacZ Tg+; ΔS50-YFP; ΔT9-CFPtriple-mutant showing that ΔT9 axons (cyan) are outside of DI (yellow) and DII (red). DII glomeruli are densely innervated by LacZ-positive axons (red). (G2) Higher magnification view of the area indicated in (G1). Scale bar = 500 μm in B–F; 120 μm in G1, 80 μm in G2.
Figure 4
Figure 4. ΔT4-YFP OSNs selectively co-express TAARs
(A) Schematic of gene choice in ΔT4-OSNs. A nascent OSN (grey) chooses the ΔT4 allele (yellow box) and is labeled (yellow cell), does not express a functional receptor (grey cilia), and makes an alternate choice (dashed arrows). (B) Epithelial section from a ΔT4-YFP mouse. OSNs expressing the ΔT4-YFP allele are labeled (green) by immunohistochemistry for YFP; TAAR-expressing OSNs are labeled (red) by in situ hybridization using a TAAR probe mix. Double-labeled OSNs (yellow) co-express ΔT4-YFP and a dorsal TAAR gene. Scale bar = 20 μm. (C) Co-expression rates for class I ORs, class II ORs, and TAARs in ΔT4-OSNs. Each class is represented by a mixture of 5 dorsally expressed probes (see Experimental Procedures). (D) Co-expression rates for TAARs in class I OR-, class II OR-, and TAAR-deletion OSNs (ΔS50, ΔM72 and ΔT4) using the probe mix for all TAARs. The higher co-expression rate for ΔT4 compared with (D) is expected given that this probe mix includes all 14 TAAR genes. (E) Co-expression rates for 37 individual OR probes and a Taar5 probe in ΔT4-OSNs. (F) Co-expression rates for individual TAAR genes inΔT4-OSNs. Co-expression was observed for all dorsal TAARs. Co-expression of ventral/broad TAAR genes was rare. The rate for Taar4 (grey bar) was measured in heterozygous (ΔT4-YFP/wt) mice, which retain one intact copy of Taar4. All data are pooled from counts using at least two animals. See also Figures S1 and S3.
Figure 5
Figure 5. Biased TAAR co-expression is not a cluster effect
(A) In vivo recombination strategy to generate the TAAR cluster deletion allele. Black polygons show location and orientation of olfactory TAAR genes. Non-olfactory Taar1 is shown in white. Two targeted mutations, aT1-YFP and S50→T9-CFP, introduce loxP sites (red triangles) into both ends of the cluster. When crossed in the presence of HPRT-Cre, F1 mice harboring the cluster deletion, ΔT2-9, can be recovered. S50 coding sequence shown as grey polygon, CFP as cyan box, YFP as yellow box. Both fluorescent markers and all olfactory TAAR genes are absent from the ΔT2-9 allele. (B) Medial views of the olfactory epithelium from a control ΔT4-YFP/wt (B1) mouse and a ΔT4-YFP/ΔT2–9 littermate (B2). OSNs expressing the ΔT4-YFP allele can be seen in the dorsal epithelium (green). There is a decrease in the number of OSNs expressing the ΔT4-YFP allele in the absence of transco -expression. Both images were scanned using the same gain settings. Blue is background autofluorescence. (C) Dorsal views of olfactory bulbs from control ΔT4-YFP/wt (C1) and ΔT4-YFP/ΔT2-9 littermate (C2) mice. The pattern of intensely labeled glomeruli (green) normally seen (C1) is severely reduced in the absence of transco -expression (C2). Both images were scanned using the same settings. (D) High magnification image of the same bulbs in (C). The image (D2) was scanned at higher gain. (E) Coronal sections of glomeruli in ΔT4-YFP/wt (E1) and a ΔT4-YFP/ΔT2–9 (E2).Glomeruli in DIII are densely innervated by ΔT4-YFP axons in ΔT4-YFP/wt mice. Only sparse innervation is seen in the absence of the trans cluster. Sections are counterstained with the nuclear label TOPRO-3 (blue). (F) Co-expression rates for all TAARs in ΔT4 -OSNs, in the presence (ΔT4/wt) or absence (ΔT4-YFP/ΔT2-9) of the wild-type TAAR cluster in trans. Co-expression is drastically reduced when the choice of TAAR alleles in trans is no longer available. (G) Co-expression rates for TAAR genes in ΔT4-OSNs when the trans cluster is absent (only alleles in cis are available). Co-expression is biased towards the genes immediately flanking the ΔT4-YFP allele. T7 and T8 indicate coding-sequence probes for Taar7a and Taar8b, which hybridize to all members of their respective subfamilies. Scale bar = 200 μm in B and D, 400 μm in C, and 30 μm in E.
Figure 6
Figure 6. Reducing the number of TAAR alleles increases the probability of T4-RFP expression
(A) Diagram of the hypothesis that there is a population of fixed size, d3-OSNs (grey neurons), that is restricted to choose among the TAAR alleles. (Left) In the presence of both copies of the TAAR cluster, each individual allele, i, has a specific probability of expression, pi. In heterozygous T4-RFP animals, a subset of d3-OSNs express the tagged allele as dictated by its probability, pT4. (Right) In the absence of the trans cluster (i.e. half the number of available TAAR alleles), the probability of choosing the remaining Taar4 allele (T4-RFP) and the number of RFP-labeled OSNs in the epithelium would double. If OSNs can choose among all ~800 dorsal OR alleles (~400 genes), the loss of 14 alleles should have a negligible effect on choice probability. (B) Wholemount views of the olfactory epithelia in T4-RFP/wt and T4-RFP/ΔT2-9 mice. Insets show higher magnification. Scale bar = 400 μm, 50 μm in insets. (C) Normalized counts of RFP-labeled OSNs in epithelial sections of T4-RFP/wt, and T4-RFP/ΔT2-9 mice. The number of T4-RFP OSNs was normalized to a dorsal class II population, M72-GFP, in the same sections. The number of OSNs expressing the T4-RFP allele nearly doubles in the absence of the trans TAAR cluster (error bars show standard deviations).
Figure 7
Figure 7. The dorsal TAAR glomeruli are selectively activated by amines
(A) In vivo glomerular imaging in a ΔT4-YFP heterozygous mouse. (Top left) Resting spH fluorescence imaged through thinned bone. Pseudocolored panels show odor-evoked changes in fluorescence (ΔF) in response to the indicated odorants. All panels are scaled to the same maximum ΔF. Dotted line approximates the edges of the left and right olfactory bulbs. Anterior is up. (B) (Left panel) Same mouse as in (A) showing YFP-labeled glomeruli (outlined in magenta). Pseudocolored images show maximum response projections for amine and non-amine odorants. Panel marked “amines-YFP” shows max amines responses with the outlined YFP-labeled region subtracted. Only small responses lateral to DIII remain. (Blood vessel-derived intrinsic signals can be seen in left anterior bulb). Amine responses include the amines in panel (A) plus 0.005% trimethylamine, 0.01% N-methylpiperidine, 0.01% triethylamine, 0.01% cyclohexylamine, 0.01% octylamine, 1% ethylenediamine. Non-amine odors include 1% propyl acetate, phenetole, isopropyl tiglate, 2-heptanone, and propionic acid. (C) Maximum response projections in two other mice showing clustering of amine-responsive glomeruli. Responses are scaled from 0–95% of the maximum ΔF within a given animal. Amine odors: 0.01% β-phenylethylamine, 0.001% isopentylamine, 0.005% trimethylamine, 0.01% N-methylpiperidine (mouse 1), plus 0.01% cyclohexylamine (mouse 2). (D) Odorant responses in another ΔT4-YFP heterozygous mouse overlaid onto the YFP signal. Greyscale image shows YFP-labeled projections in the left and right bulbs. Outline of midline and caudal bulb is indicated (dotted line). Response maps were thresholded below 16% maximum ΔF, pseudocolored and overlaid on top of the greyscale image. Responses to different amines are confined to YFP-labeled glomeruli. Lower right panel shows thresholded maximum projections indicating the locations of glomeruli responding to four amines cadaverine, trimethylamine, β-phenylethylamine and cyclohexylamine. A broadly tuned, β-phenylethylamine-responsive glomerulus appears white in overlay. Odor responses are scaled from 0–95% of the maximum ΔF for a given odorant presentation. (E) Response profiles of DIII (YFP-positive) glomeruli to a set of 6 odorants: CAD= 1% cadaverine, CHX= 0.001% cyclohexylamine, IPA= 0.001% isopentylamine, NMP= 0.01% N-methylpiperidine, PEA= 0.01% β-phenylethylamine, TMA= 0.005% trimethylamine. The area of each dot is proportional to response amplitude (ΔF). Response profiles are ordered by hierarchical clustering (Ward’s method) with groups indicated (colored shading).

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