A screen for genes expressed in the olfactory organs of Drosophila melanogaster identifies genes involved in olfactory behaviour

Background

For insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system.

Methodology/Principal Findings

We have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions.

Conclusions/Significance

We identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms.     

The sea lamprey Petromyzon marinus genome reveals the early origin of several chemosensory receptor families in the vertebrate lineage

Background  

In gnathostomes, chemosensory receptors (CR) expressed in olfactory epithelia are encoded by evolutionarily dynamic gene families encoding odorant receptors (OR), trace amine-associated receptors (TAAR), V1Rs and V2Rs. A limited number of OR-like sequences have been found in invertebrate chordate genomes. Whether these gene families arose in basal or advanced vertebrates has not been resolved because these families have not been examined systematically in agnathan genomes.     

Functional evolution of the trace amine associated receptors in mammals and the loss of TAAR1 in dogs

Background  

The trace amine associated receptor family is a diverse array of GPCRs that arose before the first vertebrates walked on land. Trace amine associated receptor 1 (TAAR1) is a wide spectrum aminergic receptor that acts as a modulator in brain monoaminergic systems. Other trace amine associated receptors appear to relate to environmental perception and show a birth-and-death pattern in mammals similar to olfactory receptors.     

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth,Amyelois transitella

Background

The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.

Methodology

By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.

Conclusion

We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.     

The source of spontaneous activity in the main olfactory bulb of the rat

Introduction

In vivo, most neurons in the main olfactory bulb exhibit robust spontaneous activity. This paper tests the hypothesis that spontaneous activity in olfactory receptor neurons drives much of the spontaneous activity in mitral and tufted cells via excitatory synapses.

Methods

Single units were recorded in vivo from the main olfactory bulb of a rat before, during, and after application of lidocaine to the olfactory nerve. The effect of lidocaine on the conduction of action potentials from the olfactory epithelium to the olfactory bulb was assessed by electrically stimulating the olfactory nerve rostral to the application site and monitoring the field potential evoked in the bulb.

Results

Lidocaine caused a significant decrease in the amplitude of the olfactory nerve evoked field potential that was recorded in the olfactory bulb. By contrast, the lidocaine block did not significantly alter the spontaneous activity of single units in the bulb, nor did it alter the field potential evoked by electrical stimulation of the lateral olfactory tract. Lidocaine block also did not change the temporal patters of action potential or their synchronization with respiration.

Conclusions

Spontaneous activity in neurons of the main olfactory bulb is not driven mainly by activity in olfactory receptor neurons despite the extensive convergence onto mitral and tufted cells. These results suggest that spontaneous activity of mitral and tufted is either an inherent property of these cells or is driven by centrifugal inputs to the bulb.     

Evolutionary Patterns and Selective Pressures of Odorant/Pheromone Receptor Gene Families in Teleost Fishes

Background

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 (K_A/K_S) in TAARs tended to be higher than those in ORs and V2Rs.

Conclusions/Significance

Frequent gene gains/losses and high K_A/K_S 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.     

Ancient genomic architecture for mammalian olfactory receptor clusters

Background  

Mammalian olfactory receptor (OR) genes reside in numerous genomic clusters of up to several dozen genes. Whole-genome sequence alignment nets of five mammals allow their comprehensive comparison, aimed at reconstructing the ancestral olfactory subgenome.     

Structural correlates of taste and smell loss in encephalitis disseminata

Background

Olfactory dysfunction in MS patients is reported in the literature. MRI of the olfactory bulb (OB) is discussed as a promising new testing method for measuring olfactory function (OF).Aim of this study was to explore reasons for and optimize the detection of olfactory dysfunction in MS patients with MRI.

Materials and Methods

OB and olfactory brain volume was assessed within 34 MS patients by manual segmentation. Olfactory function was tested using the Threshold-Discrimination-Identification-Test (TDI), gustatory function was tested using Taste Strips (TST).

Results

41% of the MS patients displayed olfactory dysfunction (8% of the control group), 16% displayed gustatory dysfunction (5% of the control group). There was a correlation between the OB volume and the number and volume of MS lesions in the olfactory brain. Olfactory brain volume correlated with the volume of lesions in the olfactory brain and the EDSS score. The TST score correlated with the number and volume of lesions in the olfactory brain.

Conclusion

The correlation between a higher number and volume of MS lesions with a decreased OB and olfactory brain volume could help to explain olfactory dysfunction.     

Phenotypes and <Emphasis Type="Italic">PRRT2</Emphasis> mutations in Chinese families with benign familial infantile epilepsy and infantile convulsions with paroxysmal choreoathetosis

Background

Mutations in the PRRT2 gene have been identified as the major cause of benign familial infantile epilepsy (BFIE), paroxysmal kinesigenic dyskinesia (PKD) and infantile convulsions with paroxysmal choreoathetosis/dyskinesias (ICCA). Here, we analyzed the phenotypes and PRRT2 mutations in Chinese families with BFIE and ICCA.

Methods

Clinical data were collected from 22 families with BFIE and eight families with ICCA. PRRT2 mutations were screened using PCR and direct sequencing.

Results

Ninety-five family members were clinically affected in the 22 BFIE families. During follow-up, two probands had one seizure induced by diarrhea at the age of two years. Thirty-one family members were affected in the eight ICCA families, including 11 individuals with benign infantile epilepsy, nine with PKD, and 11 with benign infantile epilepsy followed by PKD. Two individuals in one ICCA family had PKD or ICCA co-existing with migraine. One affected member in another ICCA family had experienced a fever-induced seizure at 7 years old. PRRT2 mutations were detected in 13 of the 22 BFIE families. The mutation c.649_650insC (p.R217PfsX8) was found in nine families. The mutations c.649delC (p.R217EfsX12) and c.904_905insG (p.D302GfsX39) were identified in three families and one family, respectively. PRRT2 mutations were identified in all eight ICCA families, including c.649_650insC (p.R217PfsX8), c.649delC (p.R217EfsX12), c.514_517delTCTG (p.S172RfsX3) and c.1023A?>?T (X341C). c.1023A?>?T is a novel mutation predicted to elongate the C-terminus of the protein by 28 residues.

Conclusions

Our data demonstrated that PRRT2 is the major causative gene of BFIE and ICCA in Chinese families. Site c.649 is a mutation hotspot: c.649_650insC is the most common mutation, and c.649delC is the second most common mutation in Chinese families with BFIE and ICCA. As far as we know, c.1023A?>?T is the first reported mutation in exon 4 of PRRT2. c.649delC was previously reported in PKD, ICCA and hemiplegic migraine families, but we further detected it in BFIE-only families. c.904_905insG was reported in an ICCA family, but we identified it in a BFIE family. c.514_517delTCTG was previously reported in a PKD family, but we identified it in an ICCA family. Migraine and febrile seizures plus could co-exist in ICCA families.

     

The dog and rat olfactory receptor repertoires

Background

Dogs and rats have a highly developed capability to detect and identify odorant molecules, even at minute concentrations. Previous analyses have shown that the olfactory receptors (ORs) that specifically bind odorant molecules are encoded by the largest gene family sequenced in mammals so far.

Results

We identified five amino acid patterns characteristic of ORs in the recently sequenced boxer dog and brown Norway rat genomes. Using these patterns, we retrieved 1,094 dog genes and 1,493 rat genes from these shotgun sequences. The retrieved sequences constitute the olfactory receptor repertoires of these two animals. Subsets of 20.3% (for the dog) and 19.5% (for the rat) of these genes were annotated as pseudogenes as they had one or several mutations interrupting their open reading frames. We performed phylogenetic studies and organized these two repertoires into classes, families and subfamilies.

Conclusion

We have established a complete or almost complete list of OR genes in the dog and the rat and have compared the sequences of these genes within and between the two species. Our results provide insight into the evolutionary development of these genes and the local amplifications that have led to the specific amplification of many subfamilies. We have also compared the human and rat ORs with the human and mouse OR repertoires.     

Organization of the olfactory system of the Indian major carp <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.): A scanning and transmission electron microscopy study

Catla catla, Labeo rohita, and Cirrhinus mrigala represent important alimentary fish in India. Their reproduction/breeding depends on seasons. Fish perceive external factors-stimuli and chemical signals through the olfactory system that plays the key role in central regulation of reproduction. However, no electron microscopy data are available on organization of olfactory components of these fish. We studied organization of the olfactory organ in male L. rohita using scanning (SEM) and transmission electron microscopy (TEM). This organ consists of olfactory epithelium, a short nerve, and olfactory bulb. The olfactory organ is ovoid in shape and consists of about 47–52 lamellae in adults and about 14–20 lamellae in fingerlings. These lamellae originate from the midline raphe. By SEM, microvillar sensory and ciliated non-sensory cells were observed in the lamellae. TEM revealed microvillar receptor cell with rough endoplasmic reticulum and Golgi apparatus towards apical end. Basal cells were present at the base of receptor cell, supporting cells were located adjacent to the olfactory receptor neurons, while epithelial cells—in the nonsensory part of olfactory epithelium. Mast, blastema, and macrophage cells were also found at the basement membrane. This work is the first publication on ultrastructural organization of the olfactory system of the Indian major carp, which provides information about morphological and ultrastructural organization of the olfactory system and opens new avenues for further investigation of chemical neuroanatomy, sensory signal processing, and neural regulation of reproduction in the Indian major carp.     

The <Emphasis Type="Italic">Juan</Emphasis> non-LTR retrotransposon in mosquitoes: genomic impact,vertical transmission and indications of recent and widespread activity

Background  

In contrast to DNA-mediated transposable elements (TEs), retrotransposons, particularly non-long terminal repeat retrotransposons (non-LTRs), are generally considered to have a much lower propensity towards horizontal transfer. Detailed studies on site-specific non-LTR families have demonstrated strict vertical transmission. More studies are needed with non-site-specific non-LTR families to determine whether strict vertical transmission is a phenomenon related to site specificity or a more general characteristic of all non-LTRs. Juan is a Jockey clade non-LTR retrotransposon first discovered in mosquitoes that is widely distributed in the mosquito family Culicidae. Being a non-site specific non-LTR, Juan offers an opportunity to further investigate the hypothesis that non-LTRs are genomic elements that are primarily vertically transmitted.     

Exome Sequencing of 47 Chinese Families with Cone-Rod Dystrophy: Mutations in 25 Known Causative Genes

Objective

The goal of this study was to identify mutations in 25 known causative genes in 47 unrelated Chinese families with cone-rod dystrophy (CORD).

Methods

Forty-seven probands from unrelated families with CORD were recruited. Genomic DNA prepared from leukocytes was analyzed by whole exome sequencing. Variants in the 25 genes were selected and then validated by Sanger sequencing.

Results

Fourteen potential pathogenic mutations, including nine novel and five known, were identified in 10 of the 47 families (21.28%). Homozygous, compound heterozygous, and hemizygous mutations were detected in three, four, or three families, respectively. The 14 mutations in the 10 families were distributed among CNGB3 (three families), PDE6C (two families), ABCA4 (one family), RPGRIP1 (one family), RPGR (two families), and CACNA1F (one family).

Conclusions

This study provides a brief view on mutation spectrum of the 25 genes in a Chinese cohort with CORD. Identification of novel mutations enriched our understanding of variations in these genes and their associated phenotypes. To our knowledge, this is the first systemic exome-sequencing analysis of all of the 25 CORD-associated genes.     

The location of olfactory receptors within olfactory epithelium is independent of odorant volatility and solubility

Background

Our objective was to study the pattern of olfactory receptor expression within the dorsal and ventral regions of the mouse olfactory epithelium. We hypothesized that olfactory receptors were distributed based on the chemical properties of their ligands: e.g. receptors for polar, hydrophilic and weakly volatile odorants would be present in the dorsal region of olfactory epithelium; while receptors for non-polar, more volatile odorants would be distributed to the ventral region. To test our hypothesis, we used micro-transplantation of cilia-enriched plasma membranes derived from dorsal or ventral regions of the olfactory epithelium into Xenopus oocytes for electrophysiological characterization against a panel of 100 odorants.

Findings

Odorants detected by ORs from the dorsal and ventral regions showed overlap in volatility and water solubility. We did not find evidence for a correlation between the solubility and volatility of odorants and the functional expression of olfactory receptors in the dorsal or ventral region of the olfactory epithelia.

Conclusions

No simple clustering or relationship between chemical properties of odorants could be associated with the different regions of the olfactory epithelium. These results suggest that the location of ORs within the epithelium is not organized based on the physico-chemical properties of their ligands.

     

Consolidation of an Olfactory Memory Trace in the Olfactory Bulb Is Required for Learning-Induced Survival of Adult-Born Neurons and Long-Term Memory

Background

It has recently been proposed that adult-born neurons in the olfactory bulb, whose survival is modulated by learning, support long-term olfactory memory. However, the mechanism used to select which adult-born neurons following learning will participate in the long-term retention of olfactory information is unknown. We addressed this question by investigating the effect of bulbar consolidation of olfactory learning on memory and neurogenesis.

Methodology/Principal Findings

Initially, we used a behavioral ecological approach using adult mice to assess the impact of consolidation on neurogenesis. Using learning paradigms in which consolidation time was varied, we showed that a spaced (across days), but not a massed (within day), learning paradigm increased survival of adult-born neurons and allowed long-term retention of the task. Subsequently, we used a pharmacological approach to block consolidation in the olfactory bulb, consisting in intrabulbar infusion of the protein synthesis inhibitor anisomycin, and found impaired learning and no increase in neurogenesis, while basic olfactory processing and the basal rate of adult-born neuron survival remained unaffected. Taken together these data indicate that survival of adult-born neurons during learning depends on consolidation processes taking place in the olfactory bulb.

Conclusion/Significance

We can thus propose a model in which consolidation processes in the olfactory bulb determine both survival of adult-born neurons and long-term olfactory memory. The finding that adult-born neuron survival during olfactory learning is governed by consolidation in the olfactory bulb strongly argues in favor of a role for bulbar adult-born neurons in supporting olfactory memory.     

Comparison of the canine and human olfactory receptor gene repertoires

Background

Olfactory receptors (ORs), the first dedicated molecules with which odorants physically interact to arouse an olfactory sensation, constitute the largest gene family in vertebrates, including around 900 genes in human and 1,500 in the mouse. Whereas dogs, like many other mammals, have a much keener olfactory potential than humans, only 21 canine OR genes have been described to date.

Results

In this study, 817 novel canine OR sequences were identified, and 640 have been characterized. Of the 661 characterized OR sequences, representing half of the canine repertoire, 18% are predicted to be pseudogenes, compared with 63% in human and 20% in mouse. Phylogenetic analysis of 403 canine OR sequences identified 51 families, and radiation-hybrid mapping of 562 showed that they are distributed on 24 dog chromosomes, in 37 distinct regions. Most of these regions constitute clusters of 2 to 124 closely linked genes. The two largest clusters (124 and 109 OR genes) are located on canine chromosomes 18 and 21. They are orthologous to human clusters located on human chromosomes 11q11-q13 and HSA11p15, containing 174 and 115 ORs respectively.

Conclusions

This study shows a strongly conserved genomic distribution of OR genes between dog and human, suggesting that OR genes evolved from a common mammalian ancestral repertoire by successive duplications. In addition, the dog repertoire appears to have expanded relative to that of humans, leading to the emergence of specific canine OR genes.

     

Inventory of the cichlid olfactory receptor gene repertoires: identification of olfactory genes with more than one coding exon

Background

To help understand the molecular mechanisms underlying the remarkable phenotypic diversity displayed by cichlids, the genome sequences of O. niloticus, P. nyererei, H. burtoni, N. brichardi and M. zebra were recently determined. Here, we present the contents of the olfactory receptor (OR) repertoires in the genomes of these five fishes.

Results

We performed an exhaustive TBLASTN search of the five cichlid genomes to identify their OR repertoires as completely as possible. We used as bait a set of ORs described in the literature. The cichlid repertoires thereby extracted contained large numbers of complete genes (O. niloticus 158; H. burtoni 90; M. zebra 102; N. brichardi 69; P. nyererei 88), a small numbers of pseudogenes and many “edge genes” corresponding to incomplete genes located at the ends of contigs. A phylogenetic tree was constructed and showed these repertoires include a large number of families and subfamilies. It also allowed the identification of a large number of OR analogues between cichlids with very high amino-acid identity (≥99%). Nearly 9% of the full-length cichlid OR genes are composed of several coding exons. This is very unusual for vertebrate OR genes. Nevertheless, the evidence is strong, and includes the donor and acceptor splice junction sequences; also, the positions of these genes in the phylogenetic tree indicate that they constitute subfamilies well apart from non-OR G protein-coupled receptor families.

Conclusions

Cichlid OR repertoires are made up of a larger number of genes and fewer pseudogenes than those in other teleosts except zebrafish. These ORs share all identified properties common to all fish ORs; however, the large number of families and subfamilies, each containing few ORs implies that they have evolved more rapidly. This high level of OR diversity is consistent with the substantial phenotypic diversity that characterizes cichlids.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-586) contains supplementary material, which is available to authorized users.     

Assessment of Olfactory Nerve by SPECT-MRI Image with Nasal Thallium-201 Administration in Patients with Olfactory Impairments in Comparison to Healthy Volunteers

Purpose

The aim of this study was to assess whether migration of thallium-201 (²⁰¹Tl) to the olfactory bulb were reduced in patients with olfactory impairments in comparison to healthy volunteers after nasal administration of ²⁰¹Tl.

Procedures

10 healthy volunteers and 21 patients enrolled in the study (19 males and 12 females; 26–71 years old). The causes of olfactory dysfunction in the patients were head trauma (n = 7), upper respiratory tract infection (n = 7), and chronic rhinosinusitis (n = 7). ²⁰¹TlCl was administered unilaterally to the olfactory cleft, and SPECT-CT was conducted 24 h later. Separate MRI images were merged with the SPECT images. ²⁰¹Tl olfactory migration was also correlated with the volume of the olfactory bulb determined from MRI images, as well as with odor recognition thresholds measured by using T&T olfactometry.

Results

Nasal ²⁰¹Tl migration to the olfactory bulb was significantly lower in the olfactory-impaired patients than in healthy volunteers. The migration of ²⁰¹Tl to the olfactory bulb was significantly correlated with odor recognition thresholds obtained with T&T olfactometry and correlated with the volume of the olfactory bulb determined from MRI images when all subjects were included.

Conclusions

Assessment of the ²⁰¹Tl migration to the olfactory bulb was the new method for the evaluation of the olfactory nerve connectivity in patients with impaired olfaction.     

Go contributes to olfactory reception in Drosophila melanogaster

Background  

Seven-transmembrane receptors typically mediate olfactory signal transduction by coupling to G-proteins. Although insect odorant receptors have seven transmembrane domains like G-protein coupled receptors, they have an inverted membrane topology and function as ligand-gated cation channels. Consequently, the involvement of cyclic nucleotides and G proteins in insect odor reception is controversial. Since the heterotrimeric G_oα subunit is expressed in Drosophila olfactory receptor neurons, we reasoned that G_o acts together with insect odorant receptor cation channels to mediate odor-induced physiological responses.