Interhemispheric asymmetry of c-Fos expression in glomeruli and the olfactory tubercle following repeated odor stimulation

Odor adaptation allows the olfactory system to regulate sensitivity to different stimulus intensities, which is essential for preventing saturation of the cell-transducing machinery and maintaining high sensitivity to persistent and repetitive odor stimuli. Although many studies have investigated the structure and mechanisms of the mammalian olfactory system that responds to chemical sensation, few studies have considered differences in neuronal activation that depend on the manner in which the olfactory system is exposed to odorants, or examined activity patterns of olfactory-related regions in the brain under different odor exposure conditions. To address these questions, we designed three different odor exposure conditions that mimicked diverse odor environments and analyzed c-Fos-expressing cells (c-Fos+ cells) in the odor columns of the olfactory bulb (OB). We then measured differences in the proportions of c-Fos-expressing cell types depending on the odor exposure condition. Surprisingly, under the specific odor condition in which the olfactory system was repeatedly exposed to the odorant for 1 min at 5-min intervals, one of the lateral odor columns and the ipsilateral hemisphere of the olfactory tubercle had more c-Fos+ cells than the other three odor columns and the contralateral hemisphere of the olfactory tubercle. However, this interhemispheric asymmetry of c-Fos expression was not observed in the anterior piriform cortex. To confirm whether the anterior olfactory nucleus pars externa (AONpE), which connects the left and right OB, contributes to this asymmetry, AONpE-lesioned mice were analyzed under the specific odor exposure condition. Asymmetric c-Fos expression was not observed in the OB or the olfactory tubercle. These data indicate that the c-Fos expression patterns of the olfactory-related regions in the brain are influenced by the odor exposure condition and that asymmetric c-Fos expression in these regions was observed under a specific odor exposure condition due to synaptic linkage via the AONpE. © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.1

A pathogen-derived metabolite induces microglial activation via odorant receptors

Microglia (MG), the principal neuroimmune sentinels in the brain, continuously sense changes in their environment and respond to invading pathogens, toxins, and cellular debris, thereby affecting neuroinflammation. Microbial pathogens produce small metabolites that influence neuroinflammation, but the molecular mechanisms that determine whether pathogen-derived small metabolites affect microglial activation of neuroinflammation remain to be elucidated. We hypothesized that odorant receptors (ORs), the largest subfamily of G protein-coupled receptors, are involved in microglial activation by pathogen-derived small metabolites. We found that MG express high levels of two mouse ORs, Olfr110 and Olfr111, which recognize a pathogenic metabolite, 2-pentylfuran, secreted by Streptococcus pneumoniae. These interactions activate MG to engage in chemotaxis, cytokine production, phagocytosis, and reactive oxygen species generation. These effects were mediated through the G(alpha s)-cyclic adenosine monophosphate-protein kinase A-extracellular signal-regulated kinase and G(beta gamma)-phospholipase C-Ca2+ pathways. Taken together, our results reveal a novel interplay between the pathogen-derived metabolite and ORs, which has major implications for our understanding of microglial activation by pathogen recognition. Database Model data are available in the PMDB database under the accession number PM0082389.N

Olfactory marker protein expression is an indicator of olfactory receptor-associated events in non-olfactory tissues.

Olfactory receptor (OR)-associated events are mediated by well-conserved components in the olfactory epithelium, including olfactory G-protein (Golf), adenylate cyclase III (ACIII), and olfactory marker protein (OMP). The expression of ORs has recently been observed in non-olfactory tissues where they are involved in monitoring extracellular chemical cues. The large number of OR genes and their sequence similarities illustrate the need to find an effective and simple way to detect non-olfactory OR-associated events. In addition, expression profiles and physiological functions of ORs in non-olfactory tissues are largely unknown. To overcome limitations associated with using OR as a target protein, this study used OMP with Golf and ACIII as targets to screen for potential OR-mediated sensing systems in non-olfactory tissues. Here, we show using western blotting, real-time PCR, and single as well as double immunoassays that ORs and OR-associated proteins are co-expressed in diverse tissues. The results of immunohistochemical analyses showed OMP (+) cells in mouse heart and in the following cells using the corresponding marker proteins c-kit, keratin 14, calcitonin, and GFAP in mouse tissues: interstitial cells of Cajal of the bladder, medullary thymic epithelial cells of the thymus, parafollicular cells of the thyroid, and Leydig cells of the testis. The expression of ORs in OMP (+) tissues was analyzed using a refined microarray analysis and validated with RT-PCR and real-time PCR. Three ORs (olfr544, olfr558, and olfr1386) were expressed in the OMP (+) cells of the bladder and thyroid as shown using a co-immunostaining method. Together, these results suggest that OMP is involved in the OR-mediated signal transduction cascade with olfactory canonical signaling components between the nervous and endocrine systems. The results further demonstrate that OMP immunohistochemical analysis is a useful tool for identifying expression of ORs, suggesting OMP expression is an indicator of potential OR-mediated chemoreception in non-olfactory systems

Olfactory receptor Olfr544 responding to azelaic acid regulates glucagon secretion in alpha-cells of mouse pancreatic islets

Olfactory receptors (ORs) are extensively expressed in olfactory as well as non-olfactory tissues. Although many OR transcripts are expressed in non-olfactory tissues, only a few studies demonstrate the functional role of ORs. Here, we verified that mouse pancreatic α-cells express potential OR-mediated downstream effectors. Moreover, high levels of mRNA for the olfactory receptors Olfr543, Olfr544, Olfr545, and Olfr1349 were expressed in α-cells as assessed using RNA-sequencing, microarray, and quantitative real-time RT-PCR analyses. Treatment with dicarboxylic acids (azelaic acid and sebacic acid) increased intracellular Ca2+ mobilization in pancreatic α-cells. The azelaic acid-induced Ca2+ response as well as glucagon secretion was concentration- and time-dependent manner. Olfr544 was expressed in α-cells, and the EC50 value of azelaic acid to Olfr544 was 19.97 μM, whereas Olfr545 did not respond to azelaic acid. Our findings demonstrate that Olfr544 responds to azelaic acid to regulate glucagon secretion through Ca2+ mobilization in α-cells of the mouse pancreatic islets, suggesting that Olfr544 may be an important therapeutic target for metabolic diseases. © 2015 Elsevier Inc.All rights reserved.1

Immunohistochemical detection of OMP in mouse non-olfactory tissues.

<p><b>(A)</b> Olfactory marker protein (OMP) staining in olfactory epithelium (OE) was used as a positive control. In the thyroid (TR), OMP (+) cells are observed between follicles (FL) and are similar to a parafollicular cell-like population. OMP (+) cells of the bladder (BL) are assumed to be in the submucosal layer. In the testis (TT), OMP signals are displayed in Leydig cell-like populations belonging to interstitial tissue (IT), and in the thymus (TM), OMP is detected in the medullary area. The asterisks indicate OMP (+) cells. <b>(B)</b> Each tissue represents samples stained in the absence of primary antibody. MU, mucosa; SML, submucosal layer; ML, muscle layer; ST, seminiferous tubule. Scale bar: 10 μm.</p

Olfactory receptor expression levels in olfactory marker protein-positive non-olfactory tissues using RT-qPCR analyses.

<p>*Values are mean fM of mRNA ± SEM. Quantitative real-time RT-PCR was used to analyze the levels of mRNA of 11 olfactory receptors (ORs) in mouse tissues. Olfactory bulb was used as a positive control for OR expression.</p><p>*OR mRNA was quantified in reference to the <i>p</i>CI-rho-olfr544 plasmid standard and normalized to eEF-2 RNA, n = 3 independent mice.</p><p>Olfactory receptor expression levels in olfactory marker protein-positive non-olfactory tissues using RT-qPCR analyses.</p

Olfactory receptors (ORs) are expressed in OMP (+) cells.

<p>Each OR is co-expressed with the olfactory marker protein (OMP) in non-olfactory tissues using co-labeling assays. A representative image is shown for each antibody combination. The left panels show each OR with Dylight 488-conjugated anti-rabbit IgG green fluorescence (A, E, I, and M). The middle panels show OMP with Cy3-conjugated anti-goat IgG red fluorescence (B, F, J, and N), and the right panels are merged images of the two individual images in the corresponding rows (C, G, K, and O). OR51E1 (mouse olfr558 ortholog; green in A) or olfr544 (green in E) is expressed with OMP (red in B and F) in the interstitial cells of Cajal (ICC) of the bladder (BL). The olfr544 (green in I) or olfr1386 (green in M) is expressed with OMP (red in J and N) in the parafollicular cells of thyroid (TR). OR (+) cells may be OMP (+) cells in each cell type of mouse tissue as evidenced by the nearly complete overlap of OR and OMP expression. Nuclei are counterstained with DAPI (blue fluorescence), except the image for olfr1386 in thyroid. OR51E1 (olfr558)/OMP (D), olfr544/OMP (H), olfr544/OMP (L), and olfr1386/OMP (P) colocalization is supported by quantitative analysis. ***, <i>P</i> < 0.0001 using unpaired <i>t</i>-test; ###, <i>P</i> = 0.0001, and ####, <i>P</i> < 0.0001 using the Mann—Whitney <i>U</i> test OMP + OR51E1 (olfr558), olfr544, or olfr1386 versus each OR or OMP alone. Results are the means ± SEM of three or four independent areas (n = 3–5 mice). Scale bar: 10 μm.</p

Co-expression of OMP with either ACIII or G_olf in non-olfactory tissues using immunohistochemical analysis.

<p>The distribution of each olfactory signaling-associated molecule, namely, olfactory marker protein (OMP), adenylate cyclase III (ACIII), and olfactory G-protein (G_olf), in non-olfactory tissues shows different expression patterns. In bladder (BL) and thymus (TM), OMP (+) cells are co-expressed with ACIII and G_olf. OMP signals expressed in the thyroid (TR) are colocalized with ACIII, but not with G_olf. In the heart (HT) and testis (TT), OMP signals are displayed alone, which is not coincident with G_olf or ACIII.</p

Validation of commercial antibodies against the olfactory receptor (OR).

<p>A heterologous OR expression system and the endogenous OR expression in olfactory epithelium (OE) was used to validate commercially available OR antibodies against specific ORs. HEK 293 cells were transiently transfected with cDNAs coding for several associated proteins (G_olf, Ric8b, and RTP1S) and ORs (olfr544, olfr1386, olfr558, olfr1496, or OR51E1 [a human olfr558 ortholog]), and immunolabeled using antibodies against both the N-terminal epitope-Rho tag (A, E, I, M, and P) and each OR-specific antibody (B, F, J, N, and Q). The olfr1386 (B) and olfr544 (F) antibody detect Lucy-Flag-Rho-olfr1386- and Rho-olfr544-constructs in the transfected cells, respectively. These results are confirmed using the anti-Rho antibody (A and E; C and G for overlay). As a positive control, both antibodies recognize unique olfactory receptor neuron (ORN) (D and H) in OE. OR51E1 and anti-Rho antibody detect Rho-OR51E1 (J and I) and Rho-olfr558 (N and M) constructs in the transfected cells and ORN (L), which is expected since they share 94% amino acid identity as orthologs. The olfr1496 is detected by anti-Rho antibody (P), but not by commercial olfr1496 antibody (Q; R for overlay), which nonspecifically recognizes all ORN (S) in OE. Scale bar = 10 μm.</p

Protein expression profiles of OMP in various mouse tissues.

<p>Double immunoassay for olfactory marker protein (OMP). <b>(A)</b> In all tissues except for kidney (KD), the results of double immunoassays demonstrate the presence of OMP. Total mouse tissue extract (100 μg for olfactory epithelium [OE] and 20 mg for KD, skeletal muscle [SM], and thymus [TM]) was immunoprecipitated (IP) with goat anti-OMP antibodies and immunoblotted with rabbit anti-OMP antibody (double immunoassay). The apparent molecular mass of OMP (arrow) is 19 kDa in OE, SM, and TM [OMP (-)], and this band is completely blocked by preincubating the OMP antibody with purified recombinant OMP [OMP (+)]. <b>(B)</b> Mouse tissue survey for OMP expression. Total tissue extract (100 μg for OE as a positive control, 3 mg for thyroid (TR), and 20 mg for all other tissues) was used for IP. * indicates immunoglobulin. LV, liver; BL, bladder; PC, pancreas; ST, stomach; DD, duodenum; TT, testis; SP, spleen; HT, heart; LG, lung.</p