Search tips
Search criteria

Results 1-25 (619053)

Clipboard (0)

Related Articles

1.  Trpc2-expressing sensory neurons in the mouse main olfactory epithelium of type B express the soluble guanylate cyclase Gucy1b2 
Chemoreception in the mouse olfactory system occurs primarily at two chemosensory epithelia in the nasal cavity: the main olfactory epithelium (MOE) and the vomeronasal epithelium. The canonical chemosensory neurons in the MOE, the olfactory sensory neurons (OSNs), express the odorant receptor (OR) gene repertoire, and depend on Adcy3 and Cnga2 for chemosensory signal transduction. The canonical chemosensory neurons in the vomeronasal epithelium, the vomeronasal sensory neurons (VSNs), express two unrelated vomeronasal receptor (VR) gene repertoires, and involve Trpc2 for chemosensory signal transduction. Recently we reported the discovery of two types of neurons in the mouse MOE that express Trcp2 in addition to Cnga2. These cell types can be distinguished at the single-cell level by expression of Adcy3: positive, type A and negative, type B. Some type A cells express OR genes. Thus far there is no specific gene or marker for type B cells, hampering further analyses such as physiological recordings. Here, we show that among MOE cells, type B cells are unique in their expression of the soluble guanylate cyclase Gucy1b2. We came across Gucy1b2 in an explorative approach based on Long Serial Analysis of Gene Expression (LongSAGE) that we applied to single red-fluorescent cells isolated from whole olfactory mucosa and vomeronasal organ of mice of a novel Trcp2-IRES-taumCherry gene-targeted strain. The generation of a novel Gucy1b2-IRES-tauGFP gene-targeted strain enabled us to visualize coalescence of axons of type B cells into glomeruli in the main olfactory bulb. Our molecular and anatomical analyses define Gucy1b2 as a marker for type B cells within the MOE. The Gucy1b2-IRES-tauGFP strain will be useful for physiological, molecular, cellular, and anatomical studies of this newly described chemosensory subsystem.
•Trpc2 + cells exist as type A and type B in the mouse main olfactory epithelium.•We find no evidence for expression of chemosensory GPCR genes in type B cells.•We identify the soluble guanylate cyclase Gucy1b2 as a marker for type B cells.•Gucy1b2-IRES-tauGFP knockin mice will be useful for physiological studies.
PMCID: PMC4396857  PMID: 25701815
Main olfactory epithelium; Vomeronasal epithelium; Cyclic-nucleotide gated channel; Trp channel; Guanylate cyclase
2.  Evolutionary Patterns and Selective Pressures of Odorant/Pheromone Receptor Gene Families in Teleost Fishes 
PLoS ONE  2008;3(12):e4083.
Teleost fishes do not have a vomeronasal organ (VNO), and their vomeronasal receptors (V1Rs, V2Rs) are expressed in the main olfactory epithelium (MOE), as are odorant receptors (ORs) and trace amine-associated receptors (TAARs). In this study, to obtain insights into the functional distinction among the four chemosensory receptor families in teleost fishes, their evolutionary patterns were examined in zebrafish, medaka, stickleback, fugu, and spotted green pufferfish.
Methodology/Principal Findings
Phylogenetic analysis revealed that many lineage-specific gene gains and losses occurred in the teleost fish TAARs, whereas only a few gene gains and losses have taken place in the teleost fish vomeronasal receptors. In addition, synonymous and nonsynonymous nucleotide substitution rate ratios (KA/KS) in TAARs tended to be higher than those in ORs and V2Rs.
Frequent gene gains/losses and high KA/KS in teleost TAARs suggest that receptors in this family are used for detecting some species-specific chemicals such as pheromones. Conversely, conserved repertoires of V1R and V2R families in teleost fishes may imply that receptors in these families perceive common odorants for teleosts, such as amino acids. Teleost ORs showed intermediate evolutionary pattern between TAARs and vomeronasal receptors. Many teleost ORs seem to be used for common odorants, but some ORs may have evolved to recognize lineage-specific odors.
PMCID: PMC2605262  PMID: 19116654
3.  Heterogeneous Sensory Innervation and Extensive Intrabulbar Connections of Olfactory Necklace Glomeruli 
PLoS ONE  2009;4(2):e4657.
The mammalian nose employs several olfactory subsystems to recognize and transduce diverse chemosensory stimuli. These subsystems differ in their anatomical position within the nasal cavity, their targets in the olfactory forebrain, and the transduction mechanisms they employ. Here we report that they can also differ in the strategies they use for stimulus coding. Necklace glomeruli are the sole main olfactory bulb (MOB) targets of an olfactory sensory neuron (OSN) subpopulation distinguished by its expression of the receptor guanylyl cyclase GC-D and the phosphodiesterase PDE2, and by its chemosensitivity to the natriuretic peptides uroguanylin and guanylin and the gas CO2. In stark contrast to the homogeneous sensory innervation of canonical MOB glomeruli from OSNs expressing the same odorant receptor (OR), we find that each necklace glomerulus of the mouse receives heterogeneous innervation from at least two distinct sensory neuron populations: one expressing GC-D and PDE2, the other expressing olfactory marker protein. In the main olfactory system it is thought that odor identity is encoded by a combinatorial strategy and represented in the MOB by a pattern of glomerular activation. This combinatorial coding scheme requires functionally homogeneous sensory inputs to individual glomeruli by OSNs expressing the same OR and displaying uniform stimulus selectivity; thus, activity in each glomerulus reflects the stimulation of a single OSN type. The heterogeneous sensory innervation of individual necklace glomeruli by multiple, functionally distinct, OSN subtypes precludes a similar combinatorial coding strategy in this olfactory subsystem.
PMCID: PMC2645502  PMID: 19247478
4.  The extremely broad odorant response profile of mouse olfactory sensory neurons expressing the odorant receptor MOR256‐17 includes trace amine‐associated receptor ligands 
The mouse olfactory system employs ~1100 G‐protein‐coupled odorant receptors (ORs). Each mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and the expressed OR determines the odorant response properties of the OSN. The broadest odorant response profile thus far demonstrated in native mouse OSNs is for OSNs that express the OR gene SR1 (also known as Olfr124 and MOR256‐3). Here we showed that the odorant responsiveness of native mouse OSNs expressing the OR gene MOR256‐17 (also known as Olfr15 and OR3) is even broader than that of OSNs expressing SR1. We investigated the electrophysiological properties of green fluorescent protein (GFP)+ OSNs in a MOR256‐17‐IRES‐tauGFP gene‐targeted mouse strain, in parallel with GFP+ OSNs in the SR1‐IRES‐tauGFP gene‐targeted mouse strain that we previously reported. Of 35 single chemical compounds belonging to distinct structural classes, MOR256‐17+ OSNs responded to 31 chemicals, compared with 10 for SR1+ OSNs. The 10 compounds that activated SR1+ OSNs also activated MOR256‐17+ OSNs. Interestingly, MOR256‐17+ OSNs were activated by three amines (cyclohexylamine, isopenthylamine, and phenylethylamine) that are typically viewed as ligands for chemosensory neurons in the main olfactory epithelium that express trace amine‐associated receptor genes, a family of 15 genes encoding G‐protein‐coupled receptors unrelated in sequence to ORs. We did not observe differences in membrane properties, indicating that the differences in odorant response profiles between the two OSN populations were due to the expressed OR. MOR256‐17+ OSNs appear to be at one extreme of odorant responsiveness among populations of OSNs expressing distinct OR genes in the mouse.
PMCID: PMC4819710  PMID: 26666691
chemoreception; electrophysiology; main olfactory epithelium; olfaction
5.  The Olfactory Transcriptomes of Mice 
PLoS Genetics  2014;10(9):e1004593.
The olfactory (OR) and vomeronasal receptor (VR) repertoires are collectively encoded by 1700 genes and pseudogenes in the mouse genome. Most OR and VR genes were identified by comparative genomic techniques and therefore, in many of those cases, only their protein coding sequences are defined. Some also lack experimental support, due in part to the similarity between them and their monogenic, cell-specific expression in olfactory tissues. Here we use deep RNA sequencing, expression microarray and quantitative RT-PCR in both the vomeronasal organ and whole olfactory mucosa to quantify their full transcriptomes in multiple male and female mice. We find evidence of expression for all VR, and almost all OR genes that are annotated as functional in the reference genome, and use the data to generate over 1100 new, multi-exonic, significantly extended receptor gene annotations. We find that OR and VR genes are neither equally nor randomly expressed, but have reproducible distributions of abundance in both tissues. The olfactory transcriptomes are only minimally different between males and females, suggesting altered gene expression at the periphery is unlikely to underpin the striking sexual dimorphism in olfactory-mediated behavior. Finally, we present evidence that hundreds of novel, putatively protein-coding genes are expressed in these highly specialized olfactory tissues, and carry out a proof-of-principle validation. Taken together, these data provide a comprehensive, quantitative catalog of the genes that mediate olfactory perception and pheromone-evoked behavior at the periphery.
Author Summary
The sense of smell in mice involves the detection of odors and pheromones by many hundreds of olfactory and vomeronasal receptors. The genes that encode these receptors account for around 5% of the whole gene catalog, but they are poorly understood because they are very similar to each other, and are thought to be turned on randomly in only a small number of cells. Here we use multiple gene expression technologies to curate and measure the activity of all the genes involved in the detection of odors and find evidence of many new ones. We show that most genes encoding olfactory and vomeronasal receptors have complex, multi-exonic structures that generate different isoforms. We find that some receptors are consistently more abundant in the nose than others, which suggests they are not turned on randomly. This may explain why mice are particularly sensitive to some odors, but less attuned to others. We find that overall males and females differ very little in gene expression, despite having altered behavioral responses to the same odors. Thus diversity in receptor expression can explain differences in odor sensitivity, but does not appear to dictate whether sex pheromones are differentially detected by males or females.
PMCID: PMC4154679  PMID: 25187969
6.  A Large-Scale Analysis of Odor Coding in the Olfactory Epithelium 
The Journal of Neuroscience  2011;31(25):9179-9191.
Mammals can perceive and discriminate myriad volatile chemicals as having a distinct odor. Odorants are initially detected by odorant receptors (ORs) on olfactory sensory neurons (OSNs) in the nose. In the mouse, each OSN expresses one of ∼1000 different OR genes. Although OSNs and their expressed ORs constitute the fundamental units of sensory input to the brain, a comprehensive understanding of how they encode odor identities is still lacking. To gain a broader and more detailed understanding of odorant recognition and odor coding at this level, we tested the responses of 3000 mouse OSNs to 125 odorants with diverse structures and perceived odors. These studies revealed extraordinary diversity, but also bias, in odorant recognition by the OSN, and thus OR, repertoire. They indicate that most OSNs are narrowly tuned to detect a subset of odorants with related structures and often related odors, but that the repertoire also includes broadly tuned components. Strikingly, the vast majority of odorants activated a unique set of OSNs, usually two or more in combination. The resulting combinatorial codes varied in size among odorants and sometimes contained both narrowly and broadly tuned components. While many OSNs recognized multiple odorants, some appeared specific for a given pheromone or other animal-associated compound, or for one or more odorants with a particular odor quality, raising the possibility that signals derived from some OSNs and ORs might elicit an innate behavior or convey a specific odor quality.
PMCID: PMC3758579  PMID: 21697369
7.  Ancient Protostome Origin of Chemosensory Ionotropic Glutamate Receptors and the Evolution of Insect Taste and Olfaction 
PLoS Genetics  2010;6(8):e1001064.
Ionotropic glutamate receptors (iGluRs) are a highly conserved family of ligand-gated ion channels present in animals, plants, and bacteria, which are best characterized for their roles in synaptic communication in vertebrate nervous systems. A variant subfamily of iGluRs, the Ionotropic Receptors (IRs), was recently identified as a new class of olfactory receptors in the fruit fly, Drosophila melanogaster, hinting at a broader function of this ion channel family in detection of environmental, as well as intercellular, chemical signals. Here, we investigate the origin and evolution of IRs by comprehensive evolutionary genomics and in situ expression analysis. In marked contrast to the insect-specific Odorant Receptor family, we show that IRs are expressed in olfactory organs across Protostomia—a major branch of the animal kingdom that encompasses arthropods, nematodes, and molluscs—indicating that they represent an ancestral protostome chemosensory receptor family. Two subfamilies of IRs are distinguished: conserved “antennal IRs,” which likely define the first olfactory receptor family of insects, and species-specific “divergent IRs,” which are expressed in peripheral and internal gustatory neurons, implicating this family in taste and food assessment. Comparative analysis of drosophilid IRs reveals the selective forces that have shaped the repertoires in flies with distinct chemosensory preferences. Examination of IR gene structure and genomic distribution suggests both non-allelic homologous recombination and retroposition contributed to the expansion of this multigene family. Together, these findings lay a foundation for functional analysis of these receptors in both neurobiological and evolutionary studies. Furthermore, this work identifies novel targets for manipulating chemosensory-driven behaviours of agricultural pests and disease vectors.
Author Summary
Ionotropic glutamate receptors (iGluRs) are a family of cell surface proteins best known for their role in allowing neurons to communicate with each other in the brain. We recently discovered a variant class of iGluRs in the fruit fly (Drosophila melanogaster), named Ionotropic Receptors (IRs), which function as olfactory receptors in its “nose,” prompting us to ask whether iGluR/IRs might have a more general function in detection of environmental chemicals. Here, we have identified families of IRs in olfactory and taste sensory organs throughout protostomes, one of the principal branches of animal life that includes snails, worms, crustaceans, and insects. Our findings suggest that this receptor family has an evolutionary ancient function in detecting odors and tastants in the external world. By comparing the repertoires of these chemosensory IRs among both closely- and distantly-related species, we have observed dynamic patterns of expansion and divergence of these receptor families in organisms occupying very different ecological niches. Notably, many of the receptors we have identified are in insects that are of significant harm to human health, such as the malaria mosquito. These proteins represent attractive targets for novel types of insect repellents to control the host-seeking behaviors of such pest species.
PMCID: PMC2924276  PMID: 20808886
8.  The activity-dependent histone variant H2BE modulates the life span of olfactory neurons 
eLife  2012;1:e00070.
We have identified a replication-independent histone variant, Hist2h2be (referred to herein as H2be), which is expressed exclusively by olfactory chemosensory neurons. Levels of H2BE are heterogeneous among olfactory neurons, but stereotyped according to the identity of the co-expressed olfactory receptor (OR). Gain- and loss-of-function experiments demonstrate that changes in H2be expression affect olfactory function and OR representation in the adult olfactory epithelium. We show that H2BE expression is reduced by sensory activity and that it promotes neuronal cell death, such that inactive olfactory neurons display higher levels of the variant and shorter life spans. Post-translational modifications (PTMs) of H2BE differ from those of the canonical H2B, consistent with a role for H2BE in altering transcription. We propose a physiological function for H2be in modulating olfactory neuron population dynamics to adapt the OR repertoire to the environment.
eLife digest
A hallmark of the nervous systems of all mammals is their capacity to undergo changes in function that are shaped by experience. This phenomenon underlies the ability of our brains to develop properly and to learn, and also enables various sensory systems—including the visual, auditory and olfactory systems—to perform optimally in diverse environments.
In most mammals, a high-functioning olfactory system is essential for carrying out tasks that are crucial for survival, such as finding food, avoiding predators and mating. In general, sensory systems have to decipher only a limited collection of stimuli, but the olfactory system must be able to process information from thousands of distinct odors that are found in a given environment and which may vary dramatically from one environment to the next. Each odor-sensing neuron in the nose of a mammal contains just one kind of odorant receptor protein, although mammalian genomes typically encode 1000 or so different kinds of receptor proteins. This suggests that it might be possible to ‘tune’ the olfactory system to a particular environment by changing the relative numbers of the different types of neurons. Indeed, it is known that the relative abundance of each type of odor-sensing neuron changes with age and experience, and that these changes might be caused by variations in the lifespans of the neurons.
Although our understanding of how these experience-dependent changes are orchestrated at the molecular level is far from complete, it is clear that adjustments in the levels of specific gene products is necessary. But how do experiences alter the levels of gene products to give rise to lasting changes in the brain? One hypothesis is that changes to a structure called chromatin are key to this process: chromatin is an assembly of DNA molecules, which are quite long, and organizing proteins, mostly proteins known as histones, that together form a compact structure that can fit inside the nucleus of a cell.
Santoro and Dulac have now discovered a previously uncharacterized protein called H2BE that is found only in the odor-sensing neurons of mice. H2BE is a variant of a protein called H2B, which is a well-known histone. They found that in odor-sensing neurons, H2BE replaces H2B to an extent that depends on the amount of activity experienced by the neuron: H2BE is nearly undetectable in highly active neurons, but almost completely replaces H2B in neurons that are inactive. Moreover, genetic manipulation showed that the deletion of H2BE significantly extended the lifespan of neurons, whereas elevated levels of H2BE shortened their lifespan. These findings reveal an extraordinary process that involves inactive odor-sensing neurons being depleted relative to active ones over time.
How does H2BE, which differs from H2B by just five amino acids, cause such dramatic changes in neuronal composition? One hint comes from evidence that these amino acids disrupt interactions between chromatin and ‘effector’ proteins, which modulate gene activity. Consistent with this, Santoro and Dulac have found that the replacement of H2B by H2BE strongly alters gene activity, although the precise mechanism by which these alterations regulate neuronal lifespans remains to be determined. Understanding this process in detail, and exploring if similar phenomena are involved in experience-dependent changes elsewhere in the nervous system, are fascinating areas of future research.
PMCID: PMC3510456  PMID: 23240083
histone; olfactory; epigenetics; Mouse
9.  Distinct Evolutionary Patterns between Chemoreceptors of 2 Vertebrate Olfactory Systems and the Differential Tuning Hypothesis 
Molecular Biology and Evolution  2008;25(8):1593-1601.
Most tetrapod vertebrates have 2 olfactory systems, the main olfactory system (MOS) and the vomeronasal system (VNS). According to the dual olfactory hypothesis, the MOS detects environmental odorants, whereas the VNS recognizes intraspecific pheromonal cues. However, this strict functional distinction has been blurred by recent reports that both systems can perceive both types of signals. Studies of a limited number of receptors suggest that MOS receptors are broadly tuned generalists, whereas VNS receptors are narrowly tuned specialists. However, whether this distinction applies to all MOS and VNS receptors remains unknown. The differential tuning hypothesis predicts that generalist MOS receptors detect an overlapping set of ligands and thus are more likely to be conserved over evolutionary time than specialist VNS receptors, which would evolve in a more lineage-specific manner. Here we test this prediction for all olfactory chemoreceptors by examining the evolutionary patterns of MOS-expressed odorant receptors (ORs) and trace amine–associated receptors (TAARs) and VNS-expressed vomeronasal type 1 receptors (V1Rs) and vomeronasal type 2 receptors (V2Rs) in 7 tetrapods (mouse, rat, dog, opossum, platypus, chicken, and frog). The phylogenies of V1Rs and V2Rs show abundant lineage-specific gene gains/losses and virtually no one-to-one orthologs between species. Opposite patterns are found for ORs and TAARs. Analysis of functional data and ligand-binding sites of ORs confirms that paralogous chemoreceptors are more likely than orthologs to have different ligands and that functional divergence between paralogous chemoreceptors is established relatively quickly following gene duplication. Together, these results strongly suggest that the functional profile of the VNS chemoreceptor repertoire evolves much faster than that of the MOS chemoreceptor repertoire and that the differential tuning hypothesis applies to the majority, if not all, of MOS and VNS receptors.
PMCID: PMC2727380  PMID: 18460446
V1R; V2R; OR; TAAR; vomeronasal; olfactory
10.  The Receptor Guanylyl Cyclase Type D (GC-D) Ligand Uroguanylin Promotes the Acquisition of Food Preferences in Mice 
Chemical Senses  2013;38(5):391-397.
Rodents rely on olfactory stimuli to communicate information between conspecifics that is critical for health and survival. For example, rodents that detect a food odor simultaneously with the social odor carbon disulfide (CS2) will acquire a preference for that food. Disruption of the chemosensory transduction cascade in CS2-sensitive olfactory sensory neurons (OSNs) that express the receptor guanylyl cyclase type D (GC-D; GC-D+ OSNs) will prevent mice from acquiring these preferences. GC-D+ OSNs also respond to the natriuretic peptide uroguanylin, which is excreted into urine and feces. We analyzed if uroguanylin could also act as a social stimulus to promote the acquisition of food preferences. We found that feces of mice that had eaten odored food, but not unodored food, promoted a strong preference for that food in mice exposed to the feces. Olfactory exploration of uroguanylin presented with a food odor similarly produced a preference that was absent when mice were exposed to the food odor alone. Finally, the acquisition of this preference was dependent on GC-D+ OSNs, as mice lacking GC-D (Gucy2d − /− mice) showed no preference for the demonstrated food. Together with our previous findings, these results demonstrate that the diverse activators of GC-D+ OSNs elicit a common behavioral result and suggest that this specialized olfactory subsystem acts as a labeled line for a type of associative olfactory learning.
PMCID: PMC3657734  PMID: 23564012
natriuretic peptide; olfaction; olfactory subsystem; social learning
11.  Solid-State, Dye-Labeled DNA Detects Volatile Compounds in the Vapor Phase 
PLoS Biology  2008;6(1):e9.
This paper demonstrates a previously unreported property of deoxyribonucleic acid—the ability of dye-labeled, solid-state DNA dried onto a surface to detect odors delivered in the vapor phase by changes in fluorescence. This property is useful for engineering systems to detect volatiles and provides a way for artificial sensors to emulate the way cross-reactive olfactory receptors respond to and encode single odorous compounds and mixtures. Recent studies show that the vertebrate olfactory receptor repertoire arises from an unusually large gene family and that the receptor types that have been tested so far show variable breadths of response. In designing biomimetic artificial noses, the challenge has been to generate a similarly large sensor repertoire that can be manufactured with exact chemical precision and reproducibility and that has the requisite combinatorial complexity to detect odors in the real world. Here we describe an approach for generating and screening large, diverse libraries of defined sensors using single-stranded, fluorescent dye–labeled DNA that has been dried onto a substrate and pulsed with brief exposures to different odors. These new solid-state DNA-based sensors are sensitive and show differential, sequence-dependent responses. Furthermore, we show that large DNA-based sensor libraries can be rapidly screened for odor response diversity using standard high-throughput microarray methods. These observations describe new properties of DNA and provide a generalized approach for producing explicitly tailored sensor arrays that can be rationally chosen for the detection of target volatiles with different chemical structures that include biologically derived odors, toxic chemicals, and explosives.
Author Summary
Biological systems can provide engineering guidance on how evolution has solved particular problems. In the context of detecting chemicals in either the aqueous or vapor phase, two general biological approaches have emerged. The first relies on individual highly specific single receptors (sensors), each tuned to detect a single molecular species—examples include the receptors that mediate pheromone detection in insects or those that function in neurotransmission. Specificity is achieved by narrow band design. The second approach is implemented by arrays of receptors with relatively broad responses. In this case, specificity emerges from a constellation of receptor types that recognizes the molecule of interest—the canonical example here is the olfactory receptors in the main olfactory system of vertebrates. Specificity is achieved by a “one chemical–many broadly responsive detectors” paradigm. While trying to mimic the enormous odor coding ability of biological olfaction in an “artificial nose,” we searched for molecules with the requisite combinatorial capacity to serve as odor detectors. Here we show that single-stranded DNA molecules tagged with a fluorescent reporter and deposited onto solid surfaces can respond to vapor phase odor pulses in a sequence-selective manner. These findings demonstrate new properties of nucleotide molecules that can be exploited in engineered odor detection devices. In addition, this broadband responsivity to small molecules should be explored as a functional aspect of DNA (and RNA) as they exist in the normal cellular milieu.
Short sequences of solid-state DNA can selectively signal their interactions with small molecules in the vapor phase. These observations have been implemented in odor sensing in an electronic "nose" and further suggest that in vivo responses to small molecules may represent new, nongenetic attributes of DNA.
PMCID: PMC2211549  PMID: 18215112
12.  The repertoire of olfactory C family G protein-coupled receptors in zebrafish: candidate chemosensory receptors for amino acids 
BMC Genomics  2006;7:309.
Vertebrate odorant receptors comprise at least three types of G protein-coupled receptors (GPCRs): the OR, V1R, and V2R/V2R-like receptors, the latter group belonging to the C family of GPCRs. These receptor families are thought to receive chemosensory information from a wide spectrum of odorant and pheromonal cues that influence critical animal behaviors such as feeding, reproduction and other social interactions.
Using genome database mining and other informatics approaches, we identified and characterized the repertoire of 54 intact "V2R-like" olfactory C family GPCRs in the zebrafish. Phylogenetic analysis – which also included a set of 34 C family GPCRs from fugu – places the fish olfactory receptors in three major groups, which are related to but clearly distinct from other C family GPCRs, including the calcium sensing receptor, metabotropic glutamate receptors, GABA-B receptor, T1R taste receptors, and the major group of V2R vomeronasal receptor families. Interestingly, an analysis of sequence conservation and selective pressure in the zebrafish receptors revealed the retention of a conserved sequence motif previously shown to be required for ligand binding in other amino acid receptors.
Based on our findings, we propose that the repertoire of zebrafish olfactory C family GPCRs has evolved to allow the detection and discrimination of a spectrum of amino acid and/or amino acid-based compounds, which are potent olfactory cues in fish. Furthermore, as the major groups of fish receptors and mammalian V2R receptors appear to have diverged significantly from a common ancestral gene(s), these receptors likely mediate chemosensation of different classes of chemical structures by their respective organisms.
PMCID: PMC1764893  PMID: 17156446
13.  The amphioxus (Branchiostoma floridae) genome contains a highly diversified set of G protein-coupled receptors 
G protein-coupled receptors (GPCRs) are one of the largest families of genes in mammals. Branchiostoma floridae (amphioxus) is one of the species most closely related species to vertebrates.
Mining and phylogenetic analysis of the amphioxus genome showed the presence of at least 664 distinct GPCRs distributed among all the main families of GPCRs; Glutamate (18), Rhodopsin (570), Adhesion (37), Frizzled (6) and Secretin (16). Surprisingly, the Adhesion GPCR repertoire in amphioxus includes receptors with many new domains not previously observed in this family. We found many Rhodopsin GPCRs from all main groups including many amine and peptide binding receptors and several previously uncharacterized expansions were also identified. This genome has however no genes coding for bitter taste receptors (TAS2), the sweet and umami (TAS1), pheromone (VR1 or VR2) or mammalian olfactory receptors.
The amphioxus genome is remarkably rich in various GPCR subtypes while the main GPCR groups known to sense exogenous substances (such as Taste 2, mammalian olfactory, nematode chemosensory, gustatory, vomeronasal and odorant receptors) in other bilateral species are absent.
PMCID: PMC2246102  PMID: 18199322
14.  Plant odorants interfere with detection of sex pheromone signals by male Heliothis virescens 
In many insects, mate finding relies on female-released sex pheromones, which have to be deciphered by the male olfactory system within an odorous background of plant volatiles present in the environment of a calling female. With respect to pheromone-mediated mate localization, plant odorants may be neutral, favorable, or disturbing. Here we examined the impact of plant odorants on detection and coding of the major sex pheromone component, (Z)-11-hexadecenal (Z11-16:Ald) in the noctuid moth Heliothis virescens. By in vivo imaging the activity in the male antennal lobe (AL), we monitored the interference at the level of olfactory sensory neurons (OSN) to illuminate mixture interactions. The results show that stimulating the male antenna with Z11-16:Ald and distinct plant-related odorants simultaneously suppressed pheromone-evoked activity in the region of the macroglomerular complex (MGC), where Z11-16:Ald-specific OSNs terminate. Based on our previous findings that antennal detection of Z11-16:Ald involves an interplay of the pheromone binding protein (PBP) HvirPBP2 and the pheromone receptor (PR) HR13, we asked if the plant odorants may interfere with any of the elements involved in pheromone detection. Using a competitive fluorescence binding assay, we found that the plant odorants neither bind to HvirPBP2 nor affect the binding of Z11-16:Ald to the protein. However, imaging experiments analyzing a cell line that expressed the receptor HR13 revealed that plant odorants significantly inhibited the Z11-16:Ald-evoked calcium responses. Together the results indicate that plant odorants can interfere with the signaling process of the major sex pheromone component at the receptor level. Consequently, it can be assumed that plant odorants in the environment may reduce the firing activity of pheromone-specific OSNs in H. virescens and thus affect mate localization.
PMCID: PMC3465774  PMID: 23060749
pheromone detection; antennal lobe; pheromone receptor; pheromone binding protein; olfaction
15.  Ultrasensitive detection of amines by a trace amine-associated receptor 
The mammalian main olfactory pathway detects volatile chemicals using two families of G protein-coupled receptors—a large repertoire of canonical odorant receptors (ORs) and a much smaller set of Trace Amine-Associated Receptors, or TAARs. The TAARs are evolutionarily conserved in vertebrates, including humans, suggesting an indispensible role in olfaction. However, little is known about the functional properties of TAARs when expressed in native olfactory sensory neurons. Here we describe experiments using gene targeting, electrophysiology and optical imaging to study the response properties of TAAR-expressing sensory neurons and their associated glomeruli in mice. We show that olfactory sensory neurons that express a subset of the TAAR repertoire are preferentially responsive to amines. In addition, neurons expressing one of two specific TAARs, TAAR3 and TAAR4, are highly sensitive and are also broadly tuned—responding to structurally diverse amines at high concentrations. Surprisingly, we find that TAAR4 is exquisitely sensitive, with apparent affinities for a preferred ligand, phenylethylamine, rivaling those seen with mammalian pheromone receptors. We provide evidence that this unprecedented sensitivity is mediated via receptor coupling to the canonical odorant transduction cascade. The data suggest that the TAARs are evolutionarily retained in the olfactory receptor repertoire to mediate high sensitivity detection of a biologically relevant class of odorous stimuli.
PMCID: PMC3711460  PMID: 23407976
16.  Amphioxus (Branchiostoma floridae) has orthologs of vertebrate odorant receptors 
A common feature of chemosensory systems is the involvement of G protein-coupled receptors (GPCRs) in the detection of environmental stimuli. Several lineages of GPCRs are involved in vertebrate olfaction, including trace amine-associated receptors, type 1 and 2 vomeronasal receptors and odorant receptors (ORs). Gene duplication and gene loss in different vertebrate lineages have lead to an enormous amount of variation in OR gene repertoire among species; some fish have fewer than 100 OR genes, while some mammals possess more than 1000. Fascinating features of the vertebrate olfactory system include allelic exclusion, where each olfactory neuron expresses only a single OR gene, and axonal guidance where neurons expressing the same receptor project axons to common glomerulae. By identifying homologous ORs in vertebrate and in non-vertebrate chordates, we hope to expose ancestral features of the chordate olfactory system that will help us to better understand the evolution of the receptors themselves and of the cellular components of the olfactory system.
We have identified 50 full-length and 11 partial ORs in Branchiostoma floridae. No ORs were identified in Ciona intestinalis. Phylogenetic analysis places the B. floridae OR genes in a monophyletic clade with the vertebrate ORs. The majority of OR genes in amphioxus are intronless and many are also tandemly arrayed in the genome. By exposing conserved amino acid motifs and testing the ability of those motifs to discriminate between ORs and non-OR GPCRs, we identified three OR-specific amino acid motifs common in cephalochordate, fish and mammalian and ORs.
Here, we show that amphioxus has orthologs of vertebrate ORs. This conclusion demonstrates that the receptors, and perhaps other components of vertebrate olfaction, evolved at least 550 million years ago. We have also identified highly conserved amino acid motifs that may be important for maintaining receptor conformation or regulating receptor activity. We anticipate that the identification of vertebrate OR orthologs in amphioxus will lead to an improved understanding of OR gene family evolution, OR gene function, and the mechanisms that control cell-specific expression, axonal guidance, signal transduction and signal integration.
PMCID: PMC2764704  PMID: 19804645
17.  Differential Muscarinic Modulation in the Olfactory Bulb 
The Journal of Neuroscience  2015;35(30):10773-10785.
Neuromodulation of olfactory circuits by acetylcholine (ACh) plays an important role in odor discrimination and learning. Early processing of chemosensory signals occurs in two functionally and anatomically distinct regions, the main and accessory olfactory bulbs (MOB and AOB), which receive extensive cholinergic input from the basal forebrain. Here, we explore the regulation of AOB and MOB circuits by ACh, and how cholinergic modulation influences olfactory-mediated behaviors in mice. Surprisingly, despite the presence of a conserved circuit, activation of muscarinic ACh receptors revealed marked differences in cholinergic modulation of output neurons: excitation in the AOB and inhibition in the MOB. Granule cells (GCs), the most abundant intrinsic neuron in the OB, also exhibited a complex muscarinic response. While GCs in the AOB were excited, MOB GCs exhibited a dual muscarinic action in the form of a hyperpolarization and an increase in excitability uncovered by cell depolarization. Furthermore, ACh influenced the input–output relationship of mitral cells in the AOB and MOB differently showing a net effect on gain in mitral cells of the MOB, but not in the AOB. Interestingly, despite the striking differences in neuromodulatory actions on output neurons, chemogenetic inhibition of cholinergic neurons produced similar perturbations in olfactory behaviors mediated by these two regions. Decreasing ACh in the OB disrupted the natural discrimination of molecularly related odors and the natural investigation of odors associated with social behaviors. Thus, the distinct neuromodulation by ACh in these circuits could underlie different solutions to the processing of general odors and semiochemicals, and the diverse olfactory behaviors they trigger.
SIGNIFICANCE STATEMENT State-dependent cholinergic modulation of brain circuits is critical for several high-level cognitive functions, including attention and memory. Here, we provide new evidence that cholinergic modulation differentially regulates two parallel circuits that process chemosensory information, the accessory and main olfactory bulb (AOB and MOB, respectively). These circuits consist of remarkably similar synaptic arrangement and neuronal types, yet cholinergic regulation produced strikingly opposing effects in output and intrinsic neurons. Despite these differences, the chemogenetic reduction of cholinergic activity in freely behaving animals disrupted odor discrimination of simple odors, and the investigation of social odors associated with behaviors signaled by the Vomeronasal system.
PMCID: PMC4518052  PMID: 26224860
accessory olfactory bulb; aggression; cholinergic; muscarinic; olfactory; social behavior
18.  Caste-Specific Expression Patterns of Immune Response and Chemosensory Related Genes in the Leaf-Cutting Ant, Atta vollenweideri 
PLoS ONE  2013;8(11):e81518.
Leaf-cutting ants are evolutionary derived social insects with elaborated division of labor and tremendous colony sizes with millions of workers. Their social organization is mainly based on olfactory communication using different pheromones and is promoted by a pronounced size-polymorphism of workers that perform different tasks within the colony. The size polymorphism and associated behaviors are correlated to distinct antennal lobe (AL) phenotypes. Two worker phenotypes differ in number of olfactory glomeruli in the AL and the presence or absence of an extremely large glomerulus (macroglomerulus), involved in trail-pheromone reception. The males' AL contains three macroglomeruli which are presumably involved in detection of sex-pheromone components. We investigated the antennal transcriptome data of all major castes (males, queens and workers) and two worker subcastes (large and tiny workers). In order to identify putative odorant receptor genes involved in pheromone detection, we identified differentially expressed odorant receptor genes (OR-genes) using custom microarrays. In total, we found 185 OR-gene fragments that are clearly related to ORs and we identified orthologs for 70 OR-genes. Among them one OR-gene differs in relative expression between the two worker subcastes by a factor of >3 and thus is a very promising candidate gene for the trail-pheromone receptor. Using the relative expression of OR-genes in males versus queens, we identified 2 candidates for sex-pheromone receptor genes in males. In addition, we identified genes from all other chemosensory related gene families (13 chemosensory protein genes, 8 odorant binding protein genes, 2 sensory-neuron membrane protein genes, 7 ionotropic receptor genes, 2 gustatory receptor genes), and we found ant-specific expansions in the chemosensory protein gene family. In addition, a large number of genes involved in immune defense exhibited differential expression across the three different castes, and some genes even between the two worker subcastes.
PMCID: PMC3829964  PMID: 24260580
19.  Hierarchical deconstruction of mouse olfactory sensory neurons: from whole mucosa to single-cell RNA-seq 
Scientific Reports  2015;5:18178.
The mouse olfactory mucosa is a complex chemosensory tissue composed of multiple cell types, neuronal and non-neuronal. We have here applied RNA-seq hierarchically, in three steps of decreasing cellular heterogeneity: starting with crude tissue samples dissected from the nose, proceeding to flow-cytometrically sorted pools of mature olfactory sensory neurons (OSNs), and finally arriving at single mature OSNs. We show that 98.9% of intact olfactory receptor (OR) genes are expressed in mature OSNs. We uncover a hitherto unknown bipartition among mature OSNs. We find that 19 of 21 single mature OSNs each express a single intact OR gene abundantly, consistent with the one neuron-one receptor rule. For the 9 single OSNs where the two alleles of the abundantly expressed OR gene exhibit single-nucleotide polymorphisms, we demonstrate that monoallelic expression of the abundantly expressed OR gene is extremely tight. The remaining two single mature OSNs lack OR gene expression but express Trpc2 and Gucy1b2. We establish these two cells as a neuronal cell type that is fundamentally distinct from canonical, OR-expressing OSNs and that is defined by the differential, higher expression of 55 genes. We propose this tiered experimental approach as a paradigm to unravel gene expression in other cellularly heterogeneous systems.
PMCID: PMC4680959  PMID: 26670777
20.  Family structure and phylogenetic analysis of odorant receptor genes in the large yellow croaker (Larimichthys crocea) 
Chemosensory receptors, which are all G-protein-coupled receptors (GPCRs), come in four types: odorant receptors (ORs), vomeronasal receptors, trace-amine associated receptors and formyl peptide receptor-like proteins. The ORs are the most important receptors for detecting a wide range of environmental chemicals in daily life. Most fish OR genes have been identified from genome databases following the completion of the genome sequencing projects of many fishes. However, it remains unclear whether these OR genes from the genome databases are actually expressed in the fish olfactory epithelium. Thus, it is necessary to clone the OR mRNAs directly from the olfactory epithelium and to examine their expression status.
Eighty-nine full-length and 22 partial OR cDNA sequences were isolated from the olfactory epithelium of the large yellow croaker, Larimichthys crocea. Bayesian phylogenetic analysis classified the vertebrate OR genes into two types, with several clades within each type, and showed that the L. crocea OR genes of each type are more closely related to those of fugu, pufferfish and stickleback than they are to those of medaka, zebrafish and frog. The reconciled tree showed 178 duplications and 129 losses. The evolutionary relationships among OR genes in these fishes accords with their evolutionary history. The fish OR genes have experienced functional divergence, and the different clades of OR genes have evolved different functions. The result of real-time PCR shows that different clades of ORs have distinct expression levels.
We have shown about 100 OR genes to be expressed in the olfactory epithelial tissues of L. crocea. The OR genes of modern fishes duplicated from their common ancestor, and were expanded over evolutionary time. The OR genes of L. crocea are closely related to those of fugu, pufferfish and stickleback, which is consistent with its evolutionary position. The different expression levels of OR genes of large yellow croaker may suggest varying roles of ORs in olfactory function.
PMCID: PMC3162931  PMID: 21834959
21.  Olfactory Receptor Patterning in a Higher Primate 
The Journal of Neuroscience  2014;34(37):12241-12252.
The mammalian olfactory system detects a plethora of environmental chemicals that are perceived as odors or stimulate instinctive behaviors. Studies using odorant receptor (OR) genes have provided insight into the molecular and organizational strategies underlying olfaction in mice. One important unanswered question, however, is whether these strategies are conserved in primates. To explore this question, we examined the macaque, a higher primate phylogenetically close to humans. Here we report that the organization of sensory inputs in the macaque nose resembles that in mouse in some respects, but not others. As in mouse, neurons with different ORs are interspersed in the macaque nose, and there are spatial zones that differ in their complement of ORs and extend axons to different domains in the olfactory bulb of the brain. However, whereas the mouse has multiple discrete band-like zones, the macaque appears to have only two broad zones. It is unclear whether the organization of OR inputs in a rodent/primate common ancestor degenerated in primates or, alternatively became more sophisticated in rodents. The mouse nose has an additional small family of chemosensory receptors, called trace amine-associated receptors (TAARs), which may detect social cues. Here we find that TAARs are also expressed in the macaque nose, suggesting that TAARs may also play a role in human olfactory perception. We further find that one human TAAR responds to rotten fish, suggesting a possible role as a sentinel to discourage ingestion of food harboring pathogenic microorganisms.
PMCID: PMC4160765  PMID: 25209267
macaque; odorant receptor; primate; trace-amine associated receptor
22.  Synchronous evolution of an odor biosynthesis pathway and behavioral response 
Current biology : CB  2012;23(1):11-20.
Rodents use olfactory cues for species-specific behaviors. For example, mice emit odors to attract mates of the same species but not competitors of closely related species. This implies rapid evolution of olfactory signaling, although odors and chemosensory receptors involved are unknown.
Here, we identify a mouse chemosignal, trimethylamine, and its olfactory receptor, trace amine-associated receptor 5 (TAAR5), to be involved in species-specific social communication. Abundant (>1,000-fold increased) and sex-dependent trimethylamine production arose de novo along the Mus lineage after divergence from Mus caroli. The two-step trimethylamine biosynthesis pathway involves synergy between commensal microflora and a sex-dependent liver enzyme, flavin-containing monooxygenase 3 (FMO3), which oxidizes trimethylamine. One key evolutionary alteration in this pathway is the recent acquisition in Mus of male-specific Fmo3 gene repression. Coincident with its evolving biosynthesis, trimethylamine evokes species-specific behaviors, attracting mice but repelling rats. Attraction to trimethylamine is abolished in TAAR5 knockout mice, and furthermore, attraction to mouse scent is impaired by enzymatic depletion of trimethylamine or TAAR5 knockout.
TAAR5 is an evolutionarily conserved olfactory receptor required for a species-specific behavior. Synchronized changes in odor biosynthesis pathways and odor-evoked behaviors could ensure species-appropriate social interactions.
PMCID: PMC3543494  PMID: 23177478
23.  Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo 
PLoS Biology  2006;4(2):e20.
Drosophila olfactory sensory neurons (OSNs) each express two odorant receptors (ORs): a divergent member of the OR family and the highly conserved, broadly expressed receptor OR83b. OR83b is essential for olfaction in vivo and enhances OR function in vitro, but the molecular mechanism by which it acts is unknown. Here we demonstrate that OR83b heterodimerizes with conventional ORs early in the endomembrane system in OSNs, couples these complexes to the conserved ciliary trafficking pathway, and is essential to maintain the OR/OR83b complex within the sensory cilia, where odor signal transduction occurs. The OR/OR83b complex is necessary and sufficient to promote functional reconstitution of odor-evoked signaling in sensory neurons that normally respond only to carbon dioxide. Unexpectedly, unlike all known vertebrate and nematode chemosensory receptors, we find that Drosophila ORs and OR83b adopt a novel membrane topology with their N-termini and the most conserved loops in the cytoplasm. These loops mediate direct association of ORs with OR83b. Our results reveal that OR83b is a universal and integral part of the functional OR in Drosophila. This atypical heteromeric and topological design appears to be an insect-specific solution for odor recognition, making the OR/OR83b complex an attractive target for the development of highly selective insect repellents to disrupt olfactory-mediated host-seeking behaviors of insect disease vectors.
This study reveals a novel membrane topology for olfactory receptors in Drosophila and details the molecular mechanisms of receptor localization at the sensory cilia.
PMCID: PMC1334387  PMID: 16402857
24.  Genome Analysis and Expression Patterns of Odorant-Binding Proteins from the Southern House Mosquito Culex pipiens quinquefasciatus 
PLoS ONE  2009;4(7):e6237.
Olfactory-based behaviors in mosquitoes are mediated by odorant-binding proteins (OBPs). They form a multigenic family involved in the peripheral events in insect olfaction, specifically the transport of odorants to membrane-bound odorant receptors. OBPs contribute to the remarkable sensitivity of the insect's olfactory system and may be involved in the selective transport of odorants.
We have employed a combination of bioinformatics and molecular approaches to identify and characterize members of the “classic” OBP family in the Southern House mosquito Culex pipiens quinquefasciatus ( = Cx. quinquefasciatus), a vector of pathogens causing several human diseases. By taking advantage of the recently released genome sequences, we have identified fifty-three putative Cx. quinquefasciatus OBP genes by Blast searches. As a first step towards their molecular characterization, expression patterns by RT-PCR revealed thirteen genes that were detected exclusively and abundantly in chemosensory tissues. No clear differences were observed in the transcripts levels of olfactory-specific OBPs between antennae of both sexes using semi-quantitative RT-PCR. Phylogenetic and comparative analysis revealed orthologous of Cx. quinquefasciatus OBPs in Anopheles gambiae and Aedes aegypti. The identification of fifty-three putative OBP genes in Cx. quinquefasciatus highlights the diversity of this family. Tissue-specificity study suggests the existence of different functional classes within the mosquito OBP family. Most genes were detected in chemosensory as well as non chemosensory tissues indicating that they might be encapsulins, but not necessarily olfactory proteins. On the other hand, thirteen “true” OBP genes were detected exclusively in olfactory tissues and might be involved specifically in the detection of “key” semiochemicals. Interestingly, in Cx. quinquefasciatus olfactory-specific OBPs belong exclusively to four distinct phylogenetic groups which are particularly well conserved among three mosquito species.
PMCID: PMC2707629  PMID: 19606229
25.  Crypt cells are involved in kin recognition in larval zebrafish 
Scientific Reports  2016;6:24590.
Zebrafish larvae imprint on visual and olfactory kin cues at day 5 and 6 postfertilization, respectively, resulting in kin recognition later in life. Exposure to non-kin cues prevents imprinting and kin recognition. Imprinting depends on MHC class II related signals and only larvae sharing MHC class II alleles can imprint on each other. Here, we analyzed which type of olfactory sensory neuron (OSN) detects kin odor. The single teleost olfactory epithelium harbors ciliated OSNs carrying OR and TAAR gene family receptors (mammals: main olfactory epithelium) and microvillous OSNs with V1R and V2R gene family receptors (mammals: vomeronasal organ). Additionally, teleosts exhibit crypt cells which possess microvilli and cilia. We used the activity marker pERK (phosphorylated extracellular signal regulated kinase) after stimulating 9 day old zebrafish larvae with either non-kin conspecific or food odor. While food odor activated both ciliated and microvillous OSNs, only the latter were activated by conspecific odor, crypt cells showed no activation to both stimuli. Then, we tested imprinted and non-imprinted larvae (full siblings) for kin odor detection. We provide the first direct evidence that crypt cells, and likely a subpopulation of microvillous OSNs, but not ciliated OSNs, play a role in detecting a kin odor related signal.
PMCID: PMC4834543  PMID: 27087508

Results 1-25 (619053)