Axonal Odorant Receptors Mediate Axon Targeting

Abstract

In mammals, odorant receptors not only detect odors but also define the target in the olfactory bulb, where sensory neurons project to give rise to the sensory map. The odorant receptor is expressed at the cilia, where it binds odorants, and at the axon terminal. The mechanism of activation and function of the odorant receptor at the axon terminal is, however, still unknown. Here, we identify phosphatidylethanolamine-binding protein 1 as a putative ligand that activates the odorant receptor at the axon terminal and affects the turning behavior of sensory axons. Genetic ablation of phosphatidylethanolamine-binding protein 1 in mice results in a strongly disturbed olfactory sensory map. Our data suggest that the odorant receptor at the axon terminal of olfactory neurons acts as an axon guidance cue that responds to molecules originating in the olfactory bulb. The dual function of the odorant receptor links specificity of odor perception and axon targeting.

Keywords: axon targeting; axonal odorant receptors; olfactory bulb; topographic map.

Graphical abstract

Figure 1

Ca²⁺ Dynamics in Olfactory Sensory Neuron (OSN) Axon Terminals and HEK293T Cells in Response to Molecules from the Olfactory Bulb (OB) Fura-2 Ca²⁺ imaging. (A and B) Embryonic rat OSN axon terminals exhibit a prompt Ca²⁺ rise upon stimulation with the third peak of size-exclusion chromatography (SEC-3) (A) and with the second peak of ionic-exchange chromatography (IEC-2) (B). (C) Summary of results (SEC-1, n = 21; SEC-2, n = 20; SEC-3, n = 31; IEC-1, n = 92; IEC-2, n = 97; IEC-3, n = 20; IEC-4, n = 20). Bars represent SEM. (D) HEK293T cells not expressing odorant receptor (OR) (pCI = empty vector; n = 76) do not exhibit Ca²⁺ response to IEC-2 or to odors (nonanedioic acid [NA]). A prompt Ca²⁺ rise is present in response to carbachol (CCH) (used as control). (E, G, and H) HEK293T cells expressing ORs exhibit Ca²⁺ rise in response to IEC-2 and the corresponding odor. OREG-vanillic acid (VA) (E; n = 72), S6-nonanedioic acid (NA) (G; n = 63), and Olfr62-2-coumaranone (CMR) (H; n = 122) are shown. (F) Sequence of pseudocolor images of HEK293T cells expressing OREG, showing the changes in [Ca²⁺] before and upon stimulation with IEC-2 and with VA. Scale bar, 20 μm. (I) Summary of results of Ca²⁺ dynamics in HEK cells. Bars represent SEM. (J) Ca²⁺ response to IEC-2 in a mouse OSN axon terminal. (K) Pseudocolor images of the mouse OSN, showing changes in [Ca²⁺] before and after IEC-2 stimulation, locally at the axon terminal (arrowhead). Scale bar, 20 μm. (L) Summary of results (n = 18). Bar represents SEM.

Figure 2

Ca²⁺ Dynamics in Rat and Mouse OSN Axon Terminals and HEK293T Cells in Response to Phosphatidylethanolamine-Binding Protein 1 (PEBP1) Fura-2 Ca²⁺ imaging. (A and B) Normalized fluorescence ratio changes (340 nm/380 nm) at the OSN axon terminals of rat in (A) and mouse in (B), upon stimulation with PEBP1. (C) Pseudocolor images of the rat OSN, showing changes in [Ca²⁺] at the axon terminal (arrowhead) before and upon application of PEBP1. Scale bar, 10 μm. (D) Summary of results. Embryonic rat OSN, n = 72; mouse OSN, n = 57. Bars represent SEM. (E) HEK293T cells not expressing odorant receptors (ORs) (pCI = empty vector) do not exhibit Ca²⁺ response to odor, e.g., Vanillic acid (VA), or to PEBP1. A prompt Ca²⁺ rise is observed in response to CCH, used as control. (F–H) Examples of Ca²⁺ dynamics in HEK293Tcells transfected with distinct ORs (F: OREG; G:P2; H:M72) and stimulated with the corresponding odor (VA; methyl salicylate [MS]) or carbachol (CCH) and with PEBP1. (I) Summary of results (pCI, n = 112; OREG, n = 47; P2, n = 70; S6, n = 112; Olfr62, n = 45; M72, n = 110). Bars represent SEM. See also Figures S2 and S6 and Tables S1 and S2.

Figure 3

Turning Response of Rat OSN Axon Terminal in Presence of Chemical Gradients (A–E) Examples of stop frames of time-lapse imaging of isolated embryonic rat OSN axon terminals at the beginning (left panel) and at the end (central panel) of pulsatile application of chemicals from a glass pipette. Composite drawings (right panel) of the turning responses of neurites during the stimulation period were made by superimposing traces to the video records of the microscopic images. White traces depict the position of the axon terminal at the beginning, although black traces indicate the trajectory of the neurites at the end of the stimulation period (~1 h). Trajectories of the axon terminal at intermediate time points during the stimulation period are indicated by traces in shades of gray. Black arrows indicate the pipette position. OSNs were stimulated with pulsatile application of (A) Ringer’s solution, (B) forskolin (FRSK), (C) odors mixture, (D) IEC-2, and (E) PEBP1. Scale bar, 20 μm. (F) Distribution of turning angles for all neurons in response to the tested stimuli (G) Summary of results, reported as mean ± SEM. (H) Turning angles (°), in response to different stimuli, reported as mean ± SEM. One-way ANOVA; Bonferroni corrected; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. See also Figure S3.

Figure 4

Expression of PEBP1 in Rat OB (A) Schematic representation of embryonic rat OB. (B) Low-magnification image of a coronal section of the rat OB labeled for PEBP1 (red). Nuclear marker DAPI (blue) is shown. High expression of PEBP1 is detected in periglomerular cells in the anterior, medial, and lateral side of embryonic rat OB. Low expression is present in the posterior side. Rat embryos, n = 5, from 4 pregnant rats. Scale bar, 50 μm. (C–F) Higher magnification images of the anterior (C), posterior (D), medial (E), and lateral (F) areas related to the section shown in (B). Scale bar, 100 μm. (G) Schematic representation of postnatal rat OB. (H) Low-magnification image of the antero-medial portion of the OB in postnatal rat (postnatal day 2 [P2]), labeled for PEBP1 (red) and DAPI (blue). Scale bar, 50 μm. (I and J) Higher magnification of the anterior (I) and medial (J) sides of the section in (H), where high expression of PEBP1 can be observed in periglomerular cells. Scale bar, 100 μm. (K) Image of the lateral and posterior side of the OB of postnatal rat (P2), labeled for PEBP1 (red) and DAPI (blue). Scale bar, 50 μm. (L and M) Higher magnification of the lateral and posterior areas of the section in (K). PEBP1 labeling is observed around the glomeruli, in the periglomerular cells, and in the lateral aspect of the OB in (L). Low expression of PEBP1 is detected in the posterior side in (M). Postnatal rat pups, P0–P4, n = 5, from 5 litters. Scale bar, 100 μm. (N) Quantification of PEBP1 expression in the OB in embryonic and postnatal rats. Bars represent SEM. One-way ANOVA; Bonferroni corrected; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. Arrows indicate PEBP1-positive cells. Yellow arrowheads and dashed green circles indicate glomeruli surrounded by low expression of PEBP1. Solid circles indicate glomeruli surrounded by high expression of PEBP1. White arrows indicate periglomerular cells expressing high level of PEBP1. A, anterior; EPL, external plexiform layer; GL, glomerular layer; L, lateral; M, medial; p, posterior. See also Figures S4, S5, and S7.

Figure 5

Expression of PEBP1 in Mouse OB Coronal sections of the mouse OB (>P40) labeled for PEBP1 (red), OMP (green), and nuclear marker DAPI (blue). (A–D) PEBP1 is highly expressed in periglomerular cells along the anterior (A), lateral (B), and medial (C) side of the OB. PEBP1 expression is barely detected in the posterior (D) side. Scale bars, 50 μm. (E–H) Higher magnification of the areas in the dashed rectangles in (A)–(D), respectively. (E) is the higher magnification of (A), (F) of (B), (G) of (C), and (H) of (D).White arrows indicate PEBP1-positive periglomerular cells. Dashed circles indicate glomeruli surrounded by low expression of PEBP1. Scale bars, 100 μm. (I) Schematic of mouse OB. Dashed rectangles indicate the position of the area included in dashed rectangles in (A)–(D) in the whole OB. (J) Staining for PEBP1 in coronal sections of the OB of PEBP1^−/− mice. PEBP1-positive cells are not present in the OB of PEBP1^−/− mice. Scale bar, 100 μm. (K) Quantification of PEBP1 expression in the OB of wild-type (WT) (n = 8) and PEBP1^−/− (n = 4) mice. Bars represent SEM. One-way ANOVA; Bonferroni corrected; one-way ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. See also Figures S4, S5, and S7.

Figure 6

Organization of Glomeruli in Control and PEBP1 Mutant Mice (A–Y) Organization of P2 and M72 glomeruli (GFP/YFP, green, A–F) revealed by immunolabeling sections of the OB with antibodies against OMP (red, G–L) and the nuclear marker DAPI (blue, M–R). Merged images are shown (S–Y). P2 axons positive for GFP and OMP coalesce to form a main homogeneous glomerulus in control P2-GFP (A and S; mice, n = 6; bulb, n = 7), in P2-GFP; PEBP1^+/− (B and T; mice, n = 4; bulb, n = 8), and in P2-GFP; PEBP1^−/− mice (C and U; mice, n = 6; bulb, n = 9). P2 axons innervate also adjacent glomeruli that result to be formed by fibers positive for GFP and OMP (e.g., expressing P2) but also by fibers positive only for OMP (e.g., expressing a different OR, heterogeneous glomeruli) in P2-GFP; PEPB1^+/− (B and T) and in P2-GFP; PEPB1^−/− mice (C and U). M72 glomeruli (D–F) are formed by OSN axons expressing YFP and positive for OMP (e.g., homogeneous glomeruli) in control M72-YFP (mice, n = 5; bulb, n = 5; D and V) and also in M72-YFP; PEPB1^+/− (mice, n = 6; bulb, n = 9; E and W) and in M72-YFP; PEPB1^−/− mice (mice, n = 6; bulb, n = 9; F and Y). Scale bar, 100 μm. White arrows indicate homogeneous glomeruli. Yellow arrowheads indicate heterogeneous glomeruli. (Z) Summary of results. One-way ANOVA; Bonferroni corrected; ^∗∗∗p < 0.001. See also Figure S8.

Figure 7

Altered Location of P2-GFP Glomeruli in PEBP1 Mutant Mice (A–D) Whole mount view of the lateral (A and C) and medial (B and D) aspect of the OB in control P2-GFP mice (A and B) and in P2-GFP; PEBP1^−/− mice (C and D). P2-GFP-expressing fibers converge to form the main glomerulus on the lateral and on the medial surface of the OB. Scale bar, 1 mm. (E) Areas of the lateral and the medial aspects of the OB in control P2-GFP, in P2-GFP; PEBP1^+/−, and in P2-GFP; PEBP1^−/− mice, respectively. Bars represent SEM. (F) Images of cleared whole bulbs of P2-GFP control (left) and in P2-GFP; PEBP1^−/− (right) mice. The position of the medial and lateral glomeruli is represented by yellow and red dots in P2 -GFP and in P2-GFP; PEBP1^−/− mice, respectively. Scale bars, 500 μm. (G) Visualization of the average position of the glomeruli displayed in relation to a representative cleared whole bulb. Pink and black dots indicate the mean location of the medial and the lateral glomeruli, respectively, in P2-GFP (pink) and in P2-GFP; PEBP1^−/− (black) mice. Relative positions in the bulb are shown (normalized values without scale). (H and I) Localization of P2-GFP medial (H) and lateral (I) glomeruli along the ventro-dorsal (V-D) and the postero-anterior (P-A) axis of the OB in P2-GFP control (mice, n = 10; bulb, n = 18), P2-GFP; PEBP1^+/− mice (mice, n = 5; bulb, n = 9), and in P2-GFP; PEBP1^−/− mice (mice, n = 8; bulb, n = 14). P2 glomeruli location along the A-P axis of the OB is significantly shifted in P2-GFP; PEBP1^−/− mice in respect to controls. Bars represent SEM. Analysis of P2-GFP glomeruli location along the A-P axis; one-way ANOVA; Bonferroni corrected; ^∗p < 0.05; ^∗∗p < 0.01. Arrowheads indicate glomeruli. V, ventral. See also Figure S8 and Videos S1 and S2.