Lucy-tag promotes expression of sea lamprey (Petromyzon marinus) olfactory receptor on cell membrane / 中国药理学通报

Aim To elucidate the effect of Lucy-tag polypeptide label on the expression of olfactory receptor membrane in sea lamprey ( Petromyzon marinus). Methods Immunocytochemis-try, double luciferase system and calcium flow detection were used to verify the effect of polypeptide label Lucy-tag on olfactory receptors of sea lamprey. Results Polypeptide label Lucy-tag promoted the membrane expression of 13 olfactory receptors out of 20 olfactory receptors, and Lucy-tag did not affect the intrinsic activity of olfactory receptors, nor did it affect the IP3 signaling pathway of G protein coupled receptor (GPCR). Conclusions It provides a favorable technique for finding the ligand of olfactory receptor and lays a good theoretical foundation for the further study of the function of the GPCR and ligand.

Expression and Localization of Olfactory Receptor AcerOr1 from Chinese Honey Bee, Apis cerana cerana (Hymenoptera, Apidae)

ABSTRACT Olfactory receptors are essential for recognition and detection of odor in honeybees. Although we have cloned and characterized the sequence of olfactory receptor AcerOr1 before, the tissue distribution and location of this gene in the nurse and the forager worker Apis cerana cerana were not very clear. To further investigate this information of AcerOr1 gene, we analyzed its expression and localization. The results showed that AcerOr1 mRNA was predominantly expressed in the antennae of nurse and forager bees, while the AcerOr1 protein was predominant in thorax, and its expression in antennae was higher in forager than in nurse. IHC revealed that AcerOr1 mainly localized in the olfactory neurons of the antennae. In addition, the staining intensity of AcerOr1 protein by IHC was consistent with the results of qRT- PCR and western blotting. The expression of AcerOr1 in non-olfactory tissues implied that, in addition to olfaction, it may involve in other physiological processes. The localization of AcerOr1 in antennae further suggests that it is involved in the olfactory system. Our research suggest that AcerOr1 may participated in perceive pheromone and odours of floral in nurse and forager bees, and also critical for the behavior of collecting, defending, cleaning between the nurse and the forager worker bees.

A influência de polimorfismos de base única na metilação de DNA em genes de receptores olfatórios / Single nucleotide polymorphisms lead to differential DNA methylation in odorant receptor genes

Os genes de receptores olfatórios (OR) pertencem a uma família de proteínas de membrana formada por cerca de 1000 genes no genoma de camundongo. Os genes OR são expressos de forma monogênica e monoalélica nos neurônios olfatórios (OSNs). No entanto, ainda não está claro o mecanismo que permite essa forma de expressão peculiar, sobretudo, qual o papel da metilação de DNA nesse processo. Nosso estudo determinou o padrão de metilação de DNA da região promotora e codificadora do gene Olfr17. Em células de epitélio olfatório (MOE) de camundongos adultos, observamos na região codificadora (CDS) do gene uma frequência de metilação em dinucleotídeos CpG 58%, enquanto que na sua região promotora ela foi bem mais baixa. Os níveis de metilação do Olfr17 em MOE de embrião (E15.5) e fígado foram similares aos observados em MOE de animais adultos. Em seguida, analisamos se a metilação de DNA pode regular a expressão gênica do Olfr17. Utilizando animais transgênicos onde os neurônios olfatórios que expressam Olfr17 também expressam GFP, pudemos selecionar neurônios olfatórios GFP+ e analisar a metilação do gene Olfr17, que está ativo nestas células. Verificamos que o padrão geral de metilação do Olfr17, tanto na região CDS como na região promotora, não se altera quando este gene está ativo. Este resultado indica que alterações na metilação do gene Olfr17 não são necessárias para que este receptor seja expresso. Finalmente, verificamos que a região promotora do gene Olfr17, de duas linhagens de camundongos diferentes, a C57BL/6 e a 129, possuem dois polimorfismos de base única (SNPs) que alteram o conteúdo CpG. Devido a estes SNPs, a linhagem 129 apresenta dois sítios CpG adicionais, inexistentes na linhagem C57BL/6. Nossas análises mostraram que estes CpGs são frequentemente metilados, o que torna o promotor do Olfr17 de 129 significativamente mais metilado que o promotor de C57BL/6. Em seguida, nós analisamos o nível de expressão no MOE dos dois alelos de Olfr17, o 129 e o C57BL/6, utilizando ensaios de RT-qPCR. Estes experimentos demonstraram que o nível de expressão do alelo 129, que possui 3 CpGs metiladas em seu promotor, é menor que o do alelo C57BL/6, que apresenta apenas uma CpG que é pouco metilada em seu promotor. Nossos resultados sugerem que as alterações na região promotora influenciam a probabilidade com que o gene OR é escolhido para ser expresso no MOE

Olfactory receptor (OR) genes belong to a large family of membrane proteins composed of 1000 genes in the mouse genome. The OR genes are expressed in the olfactory sensory neurons (OSNs) in a monogenic and monoallelic fashion. However, the mechanisms that govern OR gene expression are unclear. Here we asked whether DNA methylation plays a role in the regulation of OR gene expression. We first determined the DNA methylation pattern in the coding (CDS) and promoter regions of the odorant receptor gene Olfr17. In olfactory epithelium (MOE) cells, the CpG methylation level in the CDS is 58% but is much lower in the promoter region of the gene. In embryonic MOE (E15.5) and liver, the levels of Olfr17 DNA methylation are similar to the ones shown in adult MOE. We next analyzed whether DNA methylation is involved in Olfr17 regulation. We isolated GFP+ neurons from transgenic mice that coexpress GFP with Olfr17, and analyzed the DNA methylation pattern of the Olfr17, which is active in these cells. We found that the general methylation pattern, both, in the coding and promoter regions is not altered in the active gene. These results indicate that changes in DNA methylation are not required for the activation of Olfr17. Finally, we found that the Olfr17 promoter region from two different mouse strains, C57BL/6 and 129, has two single-nucleotide polymorphisms (SNPs) that alter the CpG content. The SNPs lead to the existence of two additional CpGs in the 129 allele, which are absent in the C57BL/6 allele. These CpGs are frequently methylated, making the 129 Olfr17 promoter significantly more methylated than the Olfr17 promoter from C57BL/6. We next performed RT-qPCR experiments to analyze the expression levels of the 129 and C57BL/6 Olfr17 alleles in the MOE. These experiments showed that the expression level of the 129 Olfr17 allele, which contains three methylated CpGs in its promoter region, is lower than the one from C57BL/6, which contains only one, undermethylated CpG, in its promoter. Our results suggest that these promoter modifications regulate the probability of the OR gene choice

Spheroid Culture of Mammalian Olfactory Receptor Neurons: Potential Applications for a Bioelectronic Nose

The olfactory system can detect many odorants with high sensitivity and selectivity based on the expression of nearly a thousand types of olfactory receptors (ORs) in olfactory receptor neurons (ORNs). These ORs have a dynamic odorant detection range and contribute to signal encoding processes in the olfactory bulb (OB). To harness the capabilities of the olfactory system and develop a biomimetic sensor, stable culture and maintenance of ORNs are required. However, in vitro monolayer culture models have several key limitations: i) short available period of cultured neurons, ii) low cultural efficiency, and iii) long-term storage challenges. This study aims to develop a technique: i) to support the spheroid culture of primary ORN precursors facilitating stable maintenance and long-term storage, and ii) to demonstrate the viability of ORN spheroid culture in developing an olfactory system mimetic bioelectronic nose. Recombinant protein (REP; TGPG[VGRGD(VGVPG)₆]₂₀WPC) was used to form the ORN spheroids. Spheroid formation enabled preservation of primary cultured ORNs without a significant decrease in viability or the expression of stemness markers for ten days. Physiological characteristics of the ORNs were verified by monitoring intracellular calcium concentration upon odorant mixture stimulation; response upon odorant stimulation were observed at least for ten days in these cultivated ORNs differentiated from spheroids. Coupling ORNs with multi electrode array (MEA) enabled the detection and discrimination of odorants by analyzing the electrical signal patterns generated following odorant stimulation. Taken together, the ORN spheroid culture process is a promising technique for the development of a bioelectronic nose and high-throughput odorant screening device.

System-Wide Expression and Function of Olfactory Receptors in Mammals

Olfactory receptors (ORs) in mammals are generally considered to function as chemosensors in the olfactory organs of animals. They are membrane proteins that traverse the cytoplasmic membrane seven times and work generally by coupling to heterotrimeric G protein. The OR is a G protein–coupled receptor that binds the guanine nucleotide-binding G(αolf) subunit and the Gβγ dimer to recognize a wide spectrum of organic compounds in accordance with its cognate ligand. Mammalian ORs were originally identified from the olfactory epithelium of rat. However, it has been recently reported that the expression of ORs is not limited to the olfactory organ. In recent decades, they have been found to be expressed in diverse organs or tissues and even tumors in mammals. In this review, the expression and expected function of olfactory receptors that exist throughout an organism's system are discussed.

Understanding the Human Sensory Conduction of Smell / 한양의대학술지

The olfactory epithelium is the main end organ for the sense of smell in humans and vertebrates. Specially differenciated neuronal cells called olfactory receptor neurons (ORNs) play a key role in the olfactory epithelium by expressing the olfactory receptors (ORs) on their apical surface membrane. The ORs are G-protein coupled receptors that transmit signals from odorants to ORNs by molecular cascades using cyclic adenosine monophosphate, calcium ions and other molecules, which result in the depolarization of ORN. Unlike other mammalian animals, only about 30% of OR genes in the human genome are expressed. The Nobel Prize was awarded to the scientists who cloned these ORs for the first time. Each ORN expresses only a single type of OR, and ORNs which express the same type of OR converge together into the same glomeruli in the olfactory bulb. A single OR recognizes multiple odorants, and a single odorant is recognized by multiple ORs with varying affinities. At the higher neurons beyond the bulb, neuronal connections are divergent. The combinatorial model of odor identification and discrimination is well established at the convergence level, but little is known about the action mechanisms of neuronal divergence for odor identification and discrimination and further study is required.

Interplay of Signaling Molecules in Olfactory Sensory Neuron toward Signal Amplification / 한양의대학술지

Over the last decades, piles of data have been accumulated to understand the olfactory sensation in every aspect, ranging from the intracellular signaling to cognitive perception. This review focuses on the ion conduction through multiple ion channels expressed in olfactory sensory neurons (OSNs) to describe how odorant binding to olfactory receptors is transduced into an electrical signal. Olfactory signal transduction and the generation of the depolarizing receptor current occur in the cilia, where the unique extraciliary environment of the nasal mucosa assists in the neuronal activation. Upon contacting with odorants, OSNs dissociate G protein-coupled receptors, initiating a signal transduction pathway that leads to firing of action potential. This signaling pathway has a unique, two step organization: a cAMP-gated Ca2+ (CNG) channel and a Ca2+-activated Cl- channel (CACC), both of which contribute to signal amplification. This transduction mechanism requires an outward-directed driving force of Cl- established by active accumulation of Cl- within the lumen of the sensory cilia. To permit Cl- accumulation, OSNs avoid the expression of the 'Chloride Sensor: WNK3', that functions as the main Cl- exclusion co-transporter in neurons of the central nervous system (CNS). Cl- accumulation provides OSNs with the driving force for the depolarization, increasing the excitatory response magnitude. This is an interesting adaptation because of the fact that the olfactory cilia reside in the mucus, outside the body, where the concentrations of ions are not as well regulated as they are in normal interstitial compartments.

Preputial gland activates olfactory receptor neurons in rat: Calcium imaging study using laser scanning confocal microscopy.

The rodent preputial gland is one of the major sources of odours and is reported to be involved in several behavioural activities. However, how the preputial gland initiates the olfactory response to manifest the effects is not known. Olfactory receptor neurons (ORNs) present in the olfactory epithelium are involved in the perception of odorant/pheromonal compounds. In the present study, the response of rat ORNs to preputial gland extract was evaluated by calcium imaging analysis. We found that some rat ORNs responded to the preputial gland extract by exhibiting an intracellular calcium response. By contrast, the ORNs did not respond at all to the foot pad extract (control). The results indicated that the substances contained in the preputial gland might interact with a type of receptor expressed in the female rat ORNs, suggested to manifest the behavioural responses, such as social and sexual interactions. This study provided the first evidence of activation of ORNs by the preputial gland extract.

Sensopercepción olfatoria: una revisión / Olfactory sensory perception

The five senses have had a fundamental importance for survival and socialization of human beings. From an evolutionary point of view the sense of smell is the oldest. This sense has a strong representation within the genome, allowing the existence of many types of receptors that allow us to capture multiple volatile odor producing molecules, sending electrical signals to higher centers to report the outside world. Several cortical areas are activated in the brain, which are interconnected to form an extensive and complex neural network, linking for example, areas involved with memory and emotions, thus giving this sense of perceptual richness. While the concept of fl avor is largely related to the sense of taste, smell provides the necessary integration with the rest of the senses and higher functions. Fully understanding the sense of smell is relevant to health professionals. Knowing the characteristics of the receptors, the transduction processes and convergence of information in the higher centers involved, we can properly detect olfactory disorders in our patients.

Effects of steroid hormone on the expression of cyclic nucleotide-gated channels of olfactory receptor neurons / 中国耳鼻咽喉头颈外科

OBJECTIVE To observe the effects of Dexamethasone on the expression of cyclic nucleotide-gated channels (CNG channels) mRNA of olfactory receptor neurons (ORNs) by real-time quantitative reverse transcription-polymerase chain reaction(RT-PCR). METHODS Forty Wistar rats were randomly divided into four groups: 24-hours Dexamethasone treated group and its control group; 2-weeks Dexamethasone treated group and its control group. Dexamethasone was injected i.p. (1 mg/kg for 24-hours group, 0.2 mg/d for 2-weeks group). Control group rats were injected with the same volume of normal saline. Real-time quantitative RT-PCR was performed to evaluate mRNA production of CNGA2 subunits. RESULTS In Dexamethasone-injected rats, the up-regulation of CNGA2 mRNA was observed in 2-weeks group(P

The Effect of Superior Cervical Ganglionectomy on Recovery of Olfaction in Induced Anosmic Mice / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: This study was undertaken to evaluate the effect of superior cervical ganglionectomy (SCG) on anosmia, which is peripherally induced in the mice. MATERIALS AND METHOD: Three groups of mice (BCF1) were studied: normal control (nasal instillation of saline, n=6); zinc sulfate group (nasal instillation of 64 mM zinc sulfate, n=25); SCG group (superior cervical ganglionectomy after nasal instillation of 64 mM zinc sulfate, n=25). Tissues of olfactory mucosa were obtained at 1, 2, 3, 4 and 7 weeks after instillation of zinc sulfate, and processed for immunohistochemistry using antisera to olfactory marker protein (OMP) to evaluate the olfactory regeneration. RESULTS: No OMP-positive cells were observed in the first two weeks after the instillation of zinc sulfate in both zinc sulfate group and the SCG group. However, the OMP-positive cells appeared first at 3 weeks after the instillation in both groups, and gradually increased in number at 4 and 7 weeks. In the SCG group, the increase of OMP-positive cells was significantly greater than those of the zinc sulfate group. The number of OMP-positive cells in the SCG group at 7 weeks was almost similar to that of the normal control group. CONCLUSION: SCG enhances regeneration of olfactory receptor cells at 3 weeks after injury. It was inferred from the above results that SCG has a significant effect on the regeneration of olfactory receptor cells and we suggest that SCG could be an effective treatment modality for olfactory dysfunction.

The Effect of Topical Steroid Nasal Instillation in Induced Anosmic Mice / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: The purpose of this study was to evaluate the effect of intranasal Mometasone furoate instillation into the nasal cavity of mice which had peripherally induced anosmia. SUBJECTS AND METHOD: Three groups of mice were studied: normal control group (nasal instillation of normal saline, n=6), Mometasone furoate non-instillation group (no treatment after nasal instillation of zinc sulfate, n=12), and Mometasone furoate instillation group (daily mometasone furoate instillation after nasal instillation of zinc sulfate, n=12). Tissues of olfactory mucosa were obtained on 1, 2, 3, 4 weeks after the instillation of zinc sulfate, and processed for immunohistochemistry using antisera to olfactory marker protein (OMP) for evaluation of olfactory regeneration. RESULTS: No OMP-positive cells were observed in the first week after the instillation of zinc sulfate in both groups. However, OMP-positive cells began appearing in the second week in both groups and gradually increased as time goes by. In the Mometasone furoate instillation group, the increase of OMP-positive cells was significantly greater than that of Mometasone furoate non-instillation group. CONCLUSION: Mometasone furoate instillation enhances regeneration of olfactory receptor cells after injury. Mometasone furoate instillation can be suggested as an effective treatment modality for olfactory dysfunction.

The Impact of Toxicants on the Olfactory System

The olfactory system is valuable in the study of some general properties of neural system and it provides as an excellent model for studying the effects of environmental toxicants on the sensory system. For example, the olfactory receptor neuron has become an important neurobiologic model system in the area of molecular and cell biology for the study of neuronal plasticity and neuronal development, including the developmental steps of cell birth and lineage, differentiation, synaptogenesis, migration, maturation, and death. The olfactory neuroepithelium is characteristic of neuron replacement and regeneration throughout life. Olfactory receptor neurons are rapidly replaced following traumatic lesions and they are the only known projection neurons with this property. Various toxicants put the olfactory system at risk for damage. Toxic agents comprise part of health hazard to human olfaction. However, the direct and indirect effects of these agents on the olfactory system are not completely understood.

Scanning electron microscopic study of the olfactory epithelium of four coldwater hillstream teleosts from Garhwal hills (India).

This contribution deals with the scanning electron microscopic surface structure of olfactory epithelium in four hillstream teleosts from the glacialfed river Alaknanda in Garhwal Himalaya (UP, India)· The closely related species—Schizothorax plagiostomus, Schizothorax richardsonii (both bottom dweller, bottom feeder, herbiomnivorous) and Schizothoraichthys progastus (column dweller, column feeder, carniomnivorous) reveal the predominance of different types of olfactory receptor cell types separately in their respective olfactory epithelium while the distinctly related species Crossocheilus latius latius with similar nature as first two (i·e· bottom dweller, bottom feeder, herbiomnivorous) displays the presence of more microvillous cells in the olfactory epithelium· Possibly, the occurrence of particular receptor cells in a fish species is related to the ecological and feeding behaviours with distinct mechanism of olfaction·