Pheromonal communication in amphibians

Pheromonal communication is widespread in salamanders and newts and may also be important in some frogs and toads. Several amphibian pheromones have been behaviorally, biochemically and molecularly identified. These pheromones are typically peptides or proteins. Study of pheromone evolution in plethodontid salamanders has revealed that courtship pheromones have been subject to continual evolutionary change, perhaps as a result of co-evolution between the pheromonal ligand and its receptor. Pheromones are detected by the vomeronasal organ and main olfactory epithelium. Chemosensory neurons express vomeronasal receptors or olfactory receptors. Frogs have relatively large numbers of vomeronasal receptors that are transcribed in both the vomeronasal organ and the main olfactory epithelium. Salamander vomeronasal receptors apparently are restricted to the vomeronasal organ. To date, no chemosensory ligands have been matched to vomeronasal receptors or olfactory receptors so it is unknown whether particular receptor types are (1) specialized for detection of pheromones versus other chemosignals, or (2) specialized for detection of volatile, nonvolatile, or water-borne chemosignals. Despite progress in understanding amphibian pheromonal communication, only a small fraction of amphibian species have been examined. Study of additional species of amphibians will indicate which traits related to pheromonal communication are evolutionarily conserved and which traits have diverged over time.     

Distinct axonal projections from two types of olfactory receptor neurons in the middle chamber epithelium of <Emphasis Type="BoldItalic">Xenopus laevis</Emphasis>

Most vertebrates have two olfactory organs, the olfactory epithelium (OE) and the vomeronasal organ. African clawed frog, Xenopus laevis, which spends their entire life in water, have three types of olfactory sensory epithelia: the OE, the middle chamber epithelium (MCE) and the vomeronasal epithelium (VNE). The axons from these epithelia project to the dorsal part of the main olfactory bulb (d-MOB), the ventral part of the MOB (v-MOB) and the accessory olfactory bulb, respectively. In the MCE, which is thought to function in water, two types of receptor neurons (RNs) are intermingled and express one of two types of G-proteins, Golf and Go, respectively. However, axonal projections from these RNs to the v-MOB are not fully understood. In this study, we examined the expression of G-proteins by immunohistochemistry to reveal the projection pattern of olfactory RNs of Xenopus laevis, especially those in the MCE. The somata of Golf- and Go-positive RNs were separately situated in the upper and lower layers of the MCE. The former were equipped with cilia and the latter with microvilli on their apical surface. These RNs are suggested to project to the rostromedial and the caudolateral regions of the v-MOB, respectively. Such segregation patterns observed in the MCE and v-MOB are also present in the OE and olfactory bulbs of most bony fish. Thus, Xenopus laevis is a very interesting model to understand the evolution of vertebrate olfactory systems because they have a primitive, fish-type olfactory system in addition to the mammalian-type olfactory system.     

Zonal organization of the mammalian main and accessory olfactory systems

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.     

Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study

Two different polyclonal antibodies were raised to synthetic peptides corresponding to distinct putative odour receptors of rat and mouse. Both antibodies selectively labelled olfactory cilia as seen with cryofixation and immunogold ultrastructural procedures. Regions of the olfactory organ where label was detected were consistent with those found at LM levels. Immunopositive cells were rare; only up to about 0.4% of these receptor cells were labelled. Despite chemical, species, and topographic differences both antibodies behaved identically in their ultrastructural labelling patterns. For both antibodies, labelling was very specific for olfactory cilia; both bound amply to the thick proximal and the thinner and long distal parts of the cilia. Dendritic knobs showed little labelling if any. Dendritic receptor cell structures below the knobs, supporting cell structures, and respiratory cilia did not immunolabel. There were no obvious differences in morphology between labelled and unlabelled receptor cells and their cilia. Labelling could be followed up to a distance of about 15 μm from the knobs along the distal parts of the cilia. When labelled cells were observed, this signal was detectable in two, sometimes three, sections taken through these cells while being consistently absent in neighbouring cells. This pattern argues strongly for the specificity of the labelling. In conclusion, very few receptor cells labelled with the antibodies to putative odour receptors. Additionally the olfactory cilia, the cellular regions that first encounter odour molecules and that are thought to transduce the odorous signal, displayed the most intense labelling with both antibodies. Consequently, the results showed these cilia as having many copies of the putative receptors. Finally, similar patterns of subcellular labelling were displayed in two different species, despite the use of different antibodies. Thus, this study provides compelling evidence that the heptahelical putative odour receptors localize in the olfactory cilia.     

Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study

Two different polyclonal antibodies were raised to synthetic peptides corresponding to distinct putative odour receptors of rat and mouse. Both antibodies selectively labelled olfactory cilia as seen with cryofixation and immunogold ultrastructural procedures. Regions of the olfactory organ where label was detected were consistent with those found at LM levels. Immunopositive cells were rare; only up to about 0.4% of these receptor cells were labelled. Despite chemical, species, and topographic differences both antibodies behaved identically in their ultrastructural labelling patterns. For both antibodies, labelling was very specific for olfactory cilia; both bound amply to the thick proximal and the thinner and long distal parts of the cilia. Dendritic knobs showed little labelling if any. Dendritic receptor cell structures below the knobs, supporting cell structures, and respiratory cilia did not immunolabel. There were no obvious differences in morphology between labelled and unlabelled receptor cells and their cilia. Labelling could be followed up to a distance of about 15 μm from the knobs along the distal parts of the cilia. When labelled cells were observed, this signal was detectable in two, sometimes three, sections taken through these cells while being consistently absent in neighbouring cells. This pattern argues strongly for the specificity of the labelling. In conclusion, very few receptor cells labelled with the antibodies to putative odour receptors. Additionally the olfactory cilia, the cellular regions that first encounter odour molecules and that are thought to transduce the odorous signal, displayed the most intense labelling with both antibodies. Consequently, the results showed these cilia as having many copies of the putative receptors. Finally, similar patterns of subcellular labelling were displayed in two different species, despite the use of different antibodies. Thus, this study provides compelling evidence that the heptahelical putative odour receptors localize in the olfactory cilia.     

Olfactory metamorphosis in the Coastal Giant Salamander (Dicamptodon tenebrosus)

This study examined the gross morphology and ultrastructure of the olfactory organ of larvae, neotenic adults, and terrestrial adults of the Coastal Giant Salamander (Dicamptodon tenebrosus). The olfactory organ of all aquatic animals (larvae and neotenes) is similar in structure, forming a tube extending from the external naris to the choana. A nonsensory vestibule leads into the main olfactory cavity. The epithelium of the main olfactory cavity is thrown into a series of transverse valleys and ridges, with at least six dorsal and nine ventral valleys lined with olfactory epithelium, and separated by ridges of respiratory epithelium. The ridges enlarge with growth, forming large flaps extending into the lumen in neotenes. The vomeronasal organ is a diverticulum off the ventrolateral side of the main olfactory cavity. In terrestrial animals, by contrast, the vestibule has been lost. The main olfactory cavity has become much broader and dorsoventrally compressed. The prominent transverse ridges are lost, although small diagonal ridges of respiratory epithelium are found in the lateral region of the ventral olfactory epithelium. The posterior and posteromedial wall of the main olfactory cavity is composed of respiratory epithelium, in contrast to the olfactory epithelium found here in aquatic forms. The vomeronasal organ remains similar to that in large larvae, but is now connected to the mouth by a groove that extends back through the choana onto the palate. Bowman's glands are present in the main olfactory cavity at all stages, but are most abundant and best developed in terrestrial adults. They are lacking in the lateral olfactory epithelium of the main olfactory cavity. At the ultrastructural level, in aquatic animals receptor cells of the main olfactory cavity can have cilia, short microvilli, a mix of the two, or long microvilli. Supporting cells are of two types: secretory supporting cells with small, electron-dense secretory granules, and ciliated supporting cells. Receptor cells of the vomeronasal organ are exclusively microvillar, but supporting cells are secretory or ciliated, as in the main olfactory cavity. After metamorphosis two distinct types of sensory epithelium occur in the main olfactory cavity. The predominant epithelium, covering most of the roof and the medial part of the floor, is characterized by supporting cells with large, electron-lucent vesicles. The epithelium on the lateral floor of the main olfactory cavity, by contrast, resembles that of aquatic animals. Both types have both microvillar and ciliated receptor cells. No important changes are noted in cell types of the vomeronasal organ after metamorphosis. A literature survey suggests that some features of the metamorphic changes described here are characteristic of all salamanders, while others appear unique to D. tenebrosus.     

Sensitivity and transduction mechanisms of responses to general odorants in turtle vomeronasal system

(a) The responses of the vomeronasal organ to general odorants in the turtle, Geoclemys reevesii, were measured by recording the accessory olfactory bulbar responses. The threshold concentrations of the vomeronasal responses to various odorants were similar to those in main olfactory bulbar responses, indicating that vomeronasal cells lacking cilia and olfactory cells having many cilia have similar sensitivities to general odorants. (b) The vomeronasal epithelium was perfused with 100 mM NaCl solution and the salt-free solution and the effects of NaCl on the vomeronasal responses to various odorants were examined. There was no essential difference between the concentration-response curves for n-amyl acetate and menthone dissolved in 100 mM NaCl solution and those dissolved in the salt-free solution in the whole concentration range examined. The ratios of the magnitudes of vomeronasal responses in the salt-free solution to those in 100 mM NaCl solution were between 1.01 and 1.10 for seven odorants tested. (c) The magnitudes of responses to the odorants were unchanged by changes in NaCl concentrations. The replacement of Na+ with organic cations such as choline+, Bis-Tris propane2+, and N-acetyl-D-glucosamine+ did not affect the magnitudes of the responses to the odorants. The Na channel blocker amiloride also did not affect the responses. (d) The vomeronasal responses were practically unchanged by changes in CaCl2 concentration. The Ca channel blockers diltiazem and verapamil did not affect the responses. (e) The replacement of Cl- with SO4(2-) did not affect the magnitudes of the vomeronasal responses. (f) The present results suggest that ion transport across the apical membranes of vomeronasal receptor cells does not contribute to the responses to odorants in the turtle.     

Olfactory receptors and signalling elements in the Grueneberg ganglion

The Grueneberg ganglion (GG) is a cluster of neurones present in the vestibule of the anterior nasal cavity. Although its function is still elusive, recent studies have shown that cells of the GG transcribe the gene encoding the olfactory marker protein (OMP) and project their axons to glomeruli of the olfactory bulb, suggesting that they may have a chemosensory function. Chemosensory responsiveness of olfactory neurones in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) is based on the expression of either odorant receptors or vomeronasal putative pheromone receptors. To scrutinize its presumptive olfactory nature, the GG was assessed for receptor expression by extensive RT-PCR analyses, leading to the identification of a distinct vomeronasal receptor which was expressed in the majority of OMP-positive GG neurones. Along with this receptor, these cells expressed the G proteins Go and Gi, both of which are also present in sensory neurones of the vomeronasal organ. Odorant receptors were expressed by very few cells during prenatal and perinatal stages; a similar number of cells expressed adenylyl cyclase type III and G(olf/s), characteristic signalling elements of the main olfactory system. The findings of the study support the notion that the GG is in fact a subunit of the complex olfactory system, comprising cells with either a VNO-like or a MOE-like phenotype. Moreover, expression of a vomeronasal receptor indicates that the GG might serve to detect pheromones.     

The histology and fine structure of the olfactory organ of the squid Lolliguncula brevis Blainville.

The olfactory organ of the squid has a thick, pseudostratified epithelium containing five morphological types of ciliated receptors. In the simplest receptors the cilia originate separately in the distal pole of the cell. All other receptors have some type of cilia filled cavity, varying from a simple pocket of cilia at the surface to a completely closed vesicle filled with cilia in cells deep in the epithelium. The receptors are compared to cells in the rhinophore of Nautilus and the olfactory organs of coleoid cephalopods. Possible functions of the olfactory organ, based on its morphology, are discussed.     

The Olfactory System in the Hagfish Myxine glutinosa

The apical part of the olfactory epithelium in Myxine glutinosa was investigated by optical and electron microscopy. This part of the epithelium consists of supporting cells and two types of olfactory receptor cells, i.e., ciliated receptor cells and microvillous receptor cells. The olfactory cilia have a 9 + 0 pattern of the microtubules, occasionally with one pair of the doublets dislocated towards the center of the cilium. Giant cilia were observed. The supporting cells bear microvilli and are rich in tonofilaments. The supporting cells also have a secretory function, their secretion consisting mainly of acid mucopolysaccharides. An asymmetrical type of desmosome was found between the olfactory receptor cells and the supporting cells.     

黑斑蛙嗅器和犁鼻器的组化及超微结构特征

嗅感受器主要感知外界环境中化学信号分子.本文采用银染、NADPH-组化染色和电镜技术来观察黑斑侧褶蛙(Petophylax nigromaculatus)的嗅器和犁鼻器的功能差异及细胞组成.银染法可对嗅上皮和犁鼻上皮的细胞进行分类及区分.其中,支持细胞胞核深染成黑色,嗅细胞胞核银染为花斑状.细胞计数显示,犁鼻上皮的嗅神经细胞含量百分比显著高于嗅上皮.组化结果显示,黑斑侧褶蛙嗅上皮和犁鼻上皮对NADPH-d表达模式差异显著,前者表达明显高于后者.电镜结果显示,黑斑侧褶蛙嗅上皮和犁鼻上皮的支持细胞由两种类型的细胞组成,分别为纤毛型和颗粒型支持细胞.     

Goα expression in the vomeronasal organ and olfactory bulb of the tammar wallaby

The vomeronasal organ (VNO) detects pheromones via 2 large families of receptors: vomeronasal receptor 1, associated with the protein Giα2, and vomeronasal receptor 2, associated with Goα. We investigated the distribution of Goα in the developing and adult VNO and adult olfactory bulb of a marsupial, the tammar wallaby. Some cells expressed Goα as early as day 5 postpartum, but by day 30, Goα expressing cells were distributed throughout the receptor epithelium of the VNO. In the adult tammar, Goα appeared to be expressed in sensory neurons whose nuclei were mostly basally located in the vomeronasal receptor epithelium. Goα expressing vomeronasal receptor cells led to all areas of the accessory olfactory bulb (AOB). The lack of regionally restricted projection of the vomeronasal receptor cell type 2 in the tammar was similar to the uniform type, with the crucial difference that the uniform type only shows expression of Giα2 and no expression of Goα. The observed Goα staining pattern suggests that the tammar may have a third accessory olfactory type that could be intermediate to the segregated and uniform types already described.     

中华须鳗嗅觉器官形态学观察

利用光学显微镜和扫描电镜观察了10尾不同体长中华须鳗嗅觉器官的结构.结果表明:中华须鳗嗅囊呈楔型;嗅囊膜和嗅囊腹面的透明膜共同围成嗅囊腔;嗅囊长径与眼径的平均比值为2.2倍;每侧嗅囊嗅板数变化范围在30～44之间;嗅板远轴端有一纤毛和嗅孔密集的舌状游离突;嗅板上皮纤毛密集,纤毛细胞表现为3种类型:纤毛感觉细胞、纤毛非感觉细胞和微绒毛感觉细胞;纤毛非感觉细胞和微绒毛细胞也出现在嗅囊壁.嗅板上大量的纤毛表明,中华须鳗嗅囊的水动力机制应属嗅板纤毛搅动型(isosmates).除观察到嗅囊壁表面有两种类型的微嵴外,还首次在嗅板上观察到一种呈荸荠状的杆状细胞.     

Localization of G protein alpha subunits and morphology of receptor neurons in olfactory and vomeronasal epithelia in Reeve's turtle, Geoclemys reevesii

Most vertebrates have two nasal epithelia: the olfactory epithelium (OE) and the vomeronasal epithelium (VNE). The apical surfaces of OE and VNE are covered with cilia and microvilli, respectively. In rodents, signal transduction pathways involve G alpha olf and G alpha i2/G alpha o in OE and VNE, respectively. Reeve's turtles (Geoclemys reevesii) live in a semiaquatic environment. The aim of this study was to investigate the localization of G proteins and the morphological characteristics of OE and VNE in Reeve's turtle. In-situ hybridization analysis revealed that both G alpha olf and G alpha o are expressed in olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs). Immunocytochemistry of G alpha olf/s and G alpha o revealed that these two G proteins were located at the apical surface, cell bodies, and axon bundles in ORNs and VRNs. Electron microscopic analysis revealed that ORNs had both cilia and microvilli on the apical surface of the same neuron, whereas VRNs had only microvilli. Moreover G alpha olf/s was located on only the cilia of OE, whereas G alpha o was not located on cilia but on microvilli. Both G alpha olf/s and G alpha o were located on microvilli of VNE. These results imply that, in Reeve's turtle, both G alpha olf/s and G alpha o function as signal transduction molecules for chemoreception in ORNs and VRNs.     

Cartilaginous torus epithelium of the vomeronasal organ of swine and sheep]

The vomeronasal organ consists of receptor cells of microvillous type, supporting and basal cells. According to their ultrastructural organization the microvillar cells are analogous to those in the main olfactory organ in the pig and have all signs of the receptor cell: microvilli at the top and centrioles in cytoplasm, as well as the central process getting off the cell body. Both in the pig and in the sheep the supporting cells contain in their apical region a number of basal bodies with cilia, getting them off. In the receptor zones of epithelium albuminous glands predominate, in the respiratory zones--mucous ones. A great amount of liquid mucus, excreted on the surface of the epithelium by numerous glands and supporting cells, apparently, facilitates adsorption and desorption of odorous molecules from the receptor cells after their stimulation. The cilia of the supporting cells probably from the stream of the vomeronasal mucus. The cartilagenous torus epithelium of the vomeronasal organ of the pig and sheep has in general a similar structural organization. This demonstrates general for Vertebrata receptor mechanisms of odorous substances, evidently connected with perception of feramones or contact olfaction.     

Olfactory receptor trafficking involves conserved regulatory steps

Olfactory receptors are difficult to functionally express in heterologous cells. They are typically retained in the endoplasmic reticulum of cells commonly used for functional expression studies and are only released to the plasma membrane in mature cells of the olfactory receptor neuron lineage. A recently developed olfactory cell line, odora, traffics olfactory receptors to the plasma membrane when differentiated. We found that undifferentiated odora cells do not traffic olfactory receptors to their surface, even though they release the receptors to the Golgi apparatus and endosomes. This behavior differs from other cell lines tested thus far. Differentiated odora cells also properly traffic vomeronasal receptors of the VN1 type, which lack sequence similarity to olfactory receptors. ODR-4, a protein that is necessary for plasma membrane trafficking of a chemosensory receptor in nematodes, facilitates trafficking of rat olfactory receptor U131 in odora and Chinese hamster ovary cells. Olfactory receptor trafficking from the endoplasmic reticulum to the plasma membrane involves at least two steps whose regulation depends on the maturation state of cells in the olfactory receptor neuron lineage. These results also indicate that some components of the regulatory mechanism are conserved.     

Tissue expression patterns identify mouse cilia genes.

In mammals, cilia are critical for development, sensation, cell signaling, sperm motility, and fluid movement. Defects in cilia are causes of several congenital syndromes, providing additional reasons to identify cilia-related genes. We hypothesized that mRNAs selectively abundant in tissues rich in highly ciliated cells encode cilia proteins. Selective abundance in olfactory epithelium, testes, vomeronasal organ, trachea, and lung proved to be an expression pattern uniquely effective in identifying documented cilia-related genes. Known and suspected cilia-related genes were statistically overrepresented among the 99 genes identified, but the majority encoded proteins of unknown function, thereby predicting new cilia-related proteins. Evidence of expression in a highly ciliated cell, the olfactory sensory neuron, exists for 73 of the genes. In situ hybridization for 17 mRNAs confirmed expression of all 17 in olfactory sensory neurons. Most were also detected in vomeronasal sensory neurons and in neighboring tissues rich in ciliated cells such as respiratory epithelium. Immunoreactivity for one of the proteins identified, Spa17, colocalized with acetylated tubulin in the cilia layer of the olfactory epithelium. In contrast, the ciliary rootlet protein, Crocc, was located in discrete structures whose position was consistent with the dendritic knobs of the olfactory sensory neurons. A compilation of >2,000 mouse genes predicted to encode cilia-related proteins revealed a strong correlation (R = 0.99) between the number of studies predicting a gene's involvement in cilia and documented evidence of such involvement, a fact that simplifies the selection of genes for further study of the physiology of cilia.     

Distinct evolutionary patterns between chemoreceptors of 2 vertebrate olfactory systems and the differential tuning hypothesis

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.     

The cell coat of the olfactory epithelium proper and vomeronasal neuroepithelium of the rat as revealed by means of the Ruthenium-red reaction

Summary The apical cell coat of the olfactory epithelium proper and the vomeronasal neuroepithelium of the rat was investigated electronmicroscopically by means of the Ruthenium-red reaction. In the olfactory epithelium proper, the cilia of receptor cells and microvilli of supporting cells possess a cell coat measuring approximately 10 nm in thickness. In the vomeronasal neuroepithelium, the apical cell coat is thicker than in the olfactory epithelium proper. On microvilli of vomeronasal receptor cells the cell coat varies in thickness from 15 to 20 nm, and on microvilli of supporting cells it measures approximately 75 nm. The functional implications of these findings are discussed.A portion of this study was presented at the 6^th European Anatomical Congress in Hamburg. This publication is dedicated to Prof. E. KlikaSupported by the Deutsche Forschungsgemeinschaft (Br 358/5-1).