Development of the Olfactory Epithelium and Nasal Glands in TMEM16A-/- and TMEM16A+/+ Mice

TMEM16A/ANO1 is a calcium-activated chloride channel expressed in several types of epithelia and involved in various physiological processes, including proliferation and development. During mouse embryonic development, the expression of TMEM16A in the olfactory epithelium is dynamic. TMEM16A is expressed at the apical surface of the entire olfactory epithelium at embryonic day E12.5 while from E16.5 its expression is restricted to a region near the transition zone with the respiratory epithelium. To investigate whether TMEM16A plays a role in the development of the mouse olfactory epithelium, we obtained the first immunohistochemistry study comparing the morphological properties of the olfactory epithelium and nasal glands in TMEM16A^-/- and TMEM16A^+/+ littermate mice. A comparison between the expression of the olfactory marker protein and adenylyl cyclase III shows that genetic ablation of TMEM16A did not seem to affect the maturation of olfactory sensory neurons and their ciliary layer. As TMEM16A is expressed at the apical part of supporting cells and in their microvilli, we used ezrin and cytokeratin 8 as markers of microvilli and cell body of supporting cells, respectively, and found that morphology and development of supporting cells were similar in TMEM16A^-/- and TMEM16A^+/+ littermate mice. The average number of supporting cells, olfactory sensory neurons, horizontal and globose basal cells were not significantly different in the two types of mice. Moreover, we also observed that the morphology of Bowman’s glands, nasal septal glands and lateral nasal glands did not change in the absence of TMEM16A. Our results indicate that the development of mouse olfactory epithelium and nasal glands does not seem to be affected by the genetic ablation of TMEM16A.     

The fine-structural distribution of G-protein receptor kinase 3, β-arrestin-2, Ca2+/calmodulin-dependent protein kinase II and phosphodiesterase PDE1C2, and a Cl−-cotransporter in rodent olfactory epithelia

The sequentially activated molecules of olfactory signal-onset are mostly concentrated in the long, thin distal parts of olfactory epithelial receptor cell cilia. Is this also true for molecules of olfactory signal-termination and -regulation? G-protein receptor kinase 3 (GRK3) supposedly aids in signal desensitization at the level of odor receptors, whereas β-arrestin-2, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and phosphodiesterase (PDE) PDE1C2 are thought to do so at the level of the adenylyl cyclase, ACIII. The Na⁺, K⁺-2Cl^?-cotransporter NKCC1 regulates Cl^?-channel activity. In an attempt to localize the subcellular sites olfactory signal-termination and -regulation we used four antibodies to GRK3, two to β-arrestin-2, five to CaMKII (one to both the α and β form, and two each specific to CaMKII α and β), two to PDE1C2, and three to Cl^?-cotransporters. Only antibodies to Cl^?-cotransporters labeled cytoplasmic compartments of, especially, supporting cells but also those of receptor cells. For all other antibodies, immunoreactivity was mostly restricted to the olfactory epithelial luminal border, confirming light microscopic studies that had shown that antibodies to GRK3, β- arrestin-2, CaMKII, and PDE1C2 labeled this region. Labeling did indeed include receptor cell cilia but occurred in microvilli of neighboring supporting cells as well. Apical parts of microvillous cells that are distinct from supporting cells, and also of ciliated respiratory cells, immunoreacted slightly with most antibodies. When peptides were available, antibody preabsorption with an excess of peptide reduced labeling intensities. Though some of the antibodies did label apices and microvilli of vomeronasal (VNO) supporting cells, none immunoreacted with VNO sensory structures.     

The lateral line canal sensory organs of the Epaulette Shark (Hemiscyllium ocellatum)

Examination of the lateral line canals in the Epaulette Shark reveals a much more differentiated sensory system than previously reported from any elasmobranch. Two main types of lateral line canals are found. In one type rounded patches of sensory epithelia are separated by elevations of the canal floor. The other type is a straight canal without restrictions and with an almost continuous sensory epithelium. In addition, we found epithelia (type A) with very long apical microvilli on the supporting cells. These microvilli reach beyond the stereovilli of the hair cells. Another type (B) of sensory epithelium has short microvilli on the supporting cells. In this latter type of epithelium the stereovilli of the hair cells are comparatively tall and reach out beyond the supporting cell microvilli.
  New hair cells are found widely in both types of sensory epithelia. These always occur as single cells, unlike those described in teleost lateral line canal sensory epithelia where new hair cells seem to form in pairs. Dying hair cells are also widespread, indicating a continuous turnover of hair cells.     

Morphometric and ultrastructural comparison of the olfactory system in elasmobranchs: The significance of structure–function relationships based on phylogeny and ecology

This study investigated the relationship between olfactory morphology, habitat occupancy, and lifestyle in 21 elasmobranch species in a phylogenetic context. Four measures of olfactory capability, that is, the number of olfactory lamellae, the surface area of the olfactory epithelium, the mass of the olfactory bulb, and the mass of the olfactory rosette were compared between individual species and groups, comprised of species with similar habitat and/or lifestyle. Statistical analyses using generalized least squares phylogenetic regression revealed that bentho‐pelagic sharks and rays possess significantly more olfactory lamellae and larger sensory epithelial surface areas than benthic species. There was no significant correlation between either olfactory bulb or rosette mass and habitat type. There was also no significant difference between the number of lamellae or the size of the sensory surface area in groups comprised of species with similar diets, that is, groups preying predominantly on crustaceans, cephalopods, echinoderms, polychaetes, molluscs, or teleosts. However, some groups had significantly larger olfactory bulb or rosette masses than others. There was little evidence to support a correlation between phylogeny and morphology, indicating that differences in olfactory capabilities are the result of functional rather than phylogenetic adaptations. All olfactory epithelia exhibited microvilli and cilia, with microvilli in both nonsensory and sensory areas, and cilia only in sensory areas. Cilia over the sensory epithelia originated from supporting cells. In contrast to teleosts, which possess ciliated and microvillous olfactory receptor types, no ciliated olfactory receptor cells were observed. This is the first comprehensive study comparing olfactory morphology to several aspects of elasmobranch ecology in a phylogenetic context. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

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.     

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.     

Postnatal changes in the ultrastructure of the rat olfactory epithelium: the supranuclear region of the supporting cells

Summary The present electron-microscopical study reports ultrastructural changes occurring in the supranuclear region of the supporting cells of the rat olfactory epithelium during the first 16 days of postnatal life. These changes are concerned with the enclosure of receptor cell dendrites and an increase in the amount of smooth endoplasmic reticulum, which has a specific distribution in the supporting cell. An increase in microvillous projections at the free cell surface is also observed. Moreover, this report demonstrates that the cytoarchitecture of the apical portion of the olfactory epithelium at the 16th day of postnatal life is similar, with respect to the relationship between supporting cells and receptor cell dendrites, to that of adult animals.     

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride- sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)- channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.      

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory epithelial surfaces of frog,ox, rat,and dog

Summary A comparison of the tight-junctions of various cell types in the nasal epithelia of frog, ox, rat and dog shows that Bowman's gland cells have lowest number of strands (4–8), whereas olfactory receptor and supporting, and ciliated respiratory cells show no conspicuous differences and have 6–11 strands. Tight-junctional strand numbers show slight species-dependent variations. In regions where three cells join (observed for receptor and respiratory cells), fracture faces show two parallel strands which fuse at certain points. These strands run perpendicularly to the rest of the tight-junctional belt, which also shows an increased number of strands (13–16) in this region.Tight-junctions of mammalian olfactory dendritic endings usually show strands composed of particles, whereas those of the other three epithelial cell types consist of continuous or discontinuous bars. Tight-junctions of dendritic endings of the frog also conform to the latter type. Differences in strand density are only slight and range from 16–27 strands/m. Small angular gap-junctions were observed only within the tight-junctions of supporting cells in the rat.     

Developmental expression of the calcium‐activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium

Calcium‐activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium‐activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 657–675, 2014     

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory structures of frog,ox, rat,and dog

Summary A comparative study using freeze-fracturing has been made of surface structures of olfactory and nasal respiratory epithelia of frog, ox, rat and dog. Special attention has been paid to cilia and microvilli present at these surfaces, although the observations include various other structures such as small intracellular vacuoles present in the olfactory receptor endings and infrequent brush cells. Within the mucus overlying the olfactory epithelium membranous vesicles, often attached to olfactory cilia, are seen. Some of these show intramembranous particle distributions similar to those of the rest of the cilia, whereas others are devoid of particles. Smooth vesicles are also found in the mucus of other types of epithelium (respiratory epithelium and Bowman's glands). The freeze-fracture morphology of intracellular secretory vacuoles present in olfactory supporting, Bowman's and respiratory glandular cells of the frog is similar in all these epithelia. Quantitative comparisons are made of the different structures of interest. When corrected for cilia which were not observed, mammalian receptor endings bear 17 cilia on average, whereas frog receptor endings have 6 cilia. The relative magnitudes of the diameters of the cilia and microvilli are, except for frog, the same for all species studied. Dimensions of other structures e.g., axons, dendrites and dendritic endings are compared in the various species. Freeze-fracture diameters are usually larger than those seen by techniques using dehydration. Dendritic ending densities range from 4.5 × 10⁶ (frog) to 8.3 × 10⁶ (dog) endings per cm². Possible sex-dependent differences are only found for these densities and dendritic ending diameters.     

Ultrastructure of cell types of the olfactory epithelium in a catfish,Heteropneustesfossilis (Bloch)

Transmission electron microscopical study of olfactory epithelium of a mud-dwelling catfish,Heteropneustes fossilis (Bloch) shows receptor, supporting, goblet and basal cells. The receptor cells are of ciliated and microvillous type. Both ciliated and microvillous receptor cells are provided with olfactory knob. The dendrite of all the receptor cells bears many longitudinally arranged microtubules. Occurrence of the rod cell and its function is quite debatable. Specialized juctional complexes between the receptor and adjacent cells are clearly noted. The supporting cells are both ciliated and nonciliated. The ciliated supporting cells are responsible for water ventilation in the olfactory chamber as well as in the inter-lamellar spaces. This facilitates better perception of odours by the receptor cells. In addition to providing mechanical support to other cells, the nonciliated supporting cells also have a secretory function which is evident from the present study. The different stages of maturity of goblet cells are well documented. The presence of white cells in the olfactory epithelium is a very rare finding.     

Ultrastructural aspects of olfactory transduction and perireceptor events

Ultrastructural/immunocytochemical studies with well defined antibodies suggest that distal segments of olfactory cilia are the main sites of early events in olfactory signal transduction. Such studies also begin to provide specifics of the cytoskeletal make-up of olfactory epithelial cells, but knowledge about relationships between cytoskeletal and transduction components is still incomplete. Probes to less well defined chemical entities, but that distinctly label olfactory cilia, supporting cell microvilli and microvilli of microvillous cells, may serve as markers for further studies on olfactory signaling. Ultrastructural/immunocytochemical studies also suggest that supporting cells help to balance the mucous environment of olfactory cilia.     

Functional Morphology of the Olfactory Organs in the Spiny Dogfish (Squalus acanthias L.) and the Small-spotted Catshark (Scyliorhinus canicula (L.))

The morphology of the olfactory organs in two sharks, the spiny dogfish and the small-spotted catshark, was studied by light microscopy and electron microscopy (TEM and SEM). The olfactory epithelium is arranged on olfactory lamellae which are provided with secondary folds. The epithelium mainly consists of microvillous receptor cells, multiciliated supporting cells and basal cells. The find of only one type of receptor cells, the microvillous type, is discussed and the condition considered a derived (apomorphic) character. The route of the water current through the olfactory organ and the different driving forces of the ventilation process are subject to discussion. In both the pelagic dogfish and the bottom-dwelling catshark the pressure difference between the incurrent and excurrent nostrils achieved by active swimming appears to be the driving force, whereas the role of the beating of the non-sensory cilia is not evident. In the bottom-dwelling catshark the ventilation of the olfactory organ is also supported by the respiratory activity.     

Diversity in the olfactory epithelium of bony fishes: Development, lamellar arrangement, sensory neuron cell types and transduction components

In this study we use a taxon-based approach to examine previous, as well as new findings on several topics pertaining to the peripheral olfactory components in teleost fishes. These topics comprise (1) the gross anatomy of the peripheral olfactory organ, including olfactory sensory neuron subtypes and their functional parameters, (2) the ultrastructure of the olfactory epithelium, and (3) recent findings regarding the development of the nasal cavity and the olfactory epithelium. The teleosts are living ray-finned fish, and include descendants of early-diverging orders (e.g., salmon), specialized descendants (e.g., goldfish and zebrafish), as well as the Acanthopterygii, numerous species with sharp bony rays, including perch, stickleback, bass and tuna. Our survey reveals that the olfactory epithelium lines a multi-lamellar olfactory rosette in many teleosts. In Acanthopterygii, there are also examples of flat, single, double or triple folded olfactory epithelia. Diverse species ventilate the olfactory chamber with a single accessory nasal sac, whereas the presence of two sacs is confined to species within the Acanthopterygii. Recent studies in salmonids and cyprinids have shown that both ciliated olfactory sensory neurons (OSNs) and microvillous OSNs respond to amino acid odorants. Bile acids stimulate ciliated OSNs, and nucleotides activate microvillous OSNs. G-protein coupled odorant receptor molecules (OR-, V1R-, and V2R-types) have been identified in several teleost species. Ciliated OSNs express the G-protein subunit G_αolf/s, which activates cyclic AMP during transduction. Localization of G protein subunits G_α0 and G_αq/11 to microvillous or crypt OSNs, varies among different species. All teleost species appear to have microvillous and ciliated OSNs. The recently discovered crypt OSN is likewise found broadly. There is surprising diversity during ontogeny. In some species, OSNs and supporting cells derive from placodal cells; in others, supporting cells develop from epithelial (skin) cells. In some, epithelial cells covering the developing olfactory epithelium degenerate, in others, these retract. Likewise, there are different mechanisms for nostril formation. We conclude that there is considerable diversity in gross anatomy and development of the peripheral olfactory organ in teleosts, yet conservation of olfactory sensory neuron morphology. There is not sufficient information to draw conclusions regarding the diversity of teleost olfactory receptors or transduction cascades.     

Degeneration and regeneration of olfactory cells induced by ZnSO4 and other chemicals

The necrotic effect of various salt solutions was tested on the catfish olfactory mucosa. Only zinc cations were able to induce an extensive degeneration of the olfactory cells. Two different modes of irrigation of the mucosa with zinc sulfate were investigated. (1) The olfactory cavity is flushed with the chemical for not more than a few seconds. At concentrations above 30 mM, the resulting damage is very reproducible, largely concentration independent and almost completely specific for the olfactory receptor cells. The non-sensory respiratory cells are unaffected, the sustentacular cells surrounding the receptor cells are affected mainly by a loss of microvilli. The olfactory receptor cells, on the contrary, start to degenerate within a few hours and by day 4 only 20% of the original receptor population remains. Division of the mucosal basal cells increases during days 3 and 4 on and day 6 olfactory receptor cells reach the bare surface of the lamella. After day 7, the receptor population reaches a level of more than 80% of its original value. Because of the absence of sustentacular processes covering the olfactory cell's knobs on day 6, it has been possible to confirm that each of the two types of olfactory receptor cells previously characterized are concentrated on each half of the mucosa. (2) The salt is maintained in contact with the tissue for several days. After this treatment most of the lamellae are irreversibly destroyed, some regeneration occurs in limited areas of the mucosa. In these small areas, indifferent respiratory cells reappear first between 20 and 35 days. It is only when the structure of the olfactory tissue is completely reorganized that the new receptor cells reappear between days 45 and 55. Regeneration is not completed before 60–65 days.     

Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat

We used cytochrome oxidase (CytOx) staining intensity, which is correlated with neuronal functional activity, to evaluate maturity and functionality of newborn rat olfactory epithelium (OE) and olfactory receptor neurons (ORNs). Nasal olfactory tissue of neonatal rats was stained with CytOx and analyzed qualitatively and quantitatively. Results revealed that newborn OE shows six differentially stained horizontal bands. Bands run parallel to the OE surface and were categorized as very light, medium or darkly stained. A narrow and pale Band 1 overlapped with horizontal basal cells. Next, a wide and lightly stained Band 2 was observed that coincides with the globose basal cell layer and immature ORNs, deep in OE. Next apically, a medium-staining Band 3 overlapped with ORN perikarya. Closer to the surface, a medium to light Band 4 was discerned where dendrites of mature ORNs normally occur. This band was interrupted with lighter areas due to the presence of supporting cells nuclei. Next, a superficial but dark Band 5 occurred, populated by the apical portions of ORN dendrites and their ciliated knobs and by supporting cell apices; mitochondria in apices of supporting cells contribute predominantly to dense staining of this Band 5. Apical to Band 5, a thin and fairly light Band 6 was observed which overlaps with the mucus layer that contains part of the ORN knobs, their cilia and supporting cell microvilli. Along the length of ORN dendrites, apical segments just below the ORN knobs, and wide basal segments showed a darker staining than the middle segments implying “microzones” of higher neural activity within the most apical and basal regions of dendrites. Our findings agree with ultrastructural studies showing a presence of mitochondria in knobs, basal portions of ORN dendrites and in OE supporting cell apices, suggesting that apical regions of both olfactory and supporting cells near the surfaces are metabolically most active, in odorant detection, signal processing, and detoxification, the latter for supporting cells.     

Ultrastructure of the Olfactory Epithelium in the Australian Lungfish Neoceratodus forsteri

Four cell types are present in the olfactory epithelium of Neoceratodus forsteri, i.e., olfactory receptor cells, supporting cells, non-sensory ciliated cells, and basal cells. Only microvilli and no cilia were observed on the receptor cells. The neurotubules pass out into these microvilli. Conspicuous arrays of agranular endoplasmic reticulum are present in the nuclear region of the receptor cells. The supporting cells are provided with microvilli. These cells may be secretory. The non-sensory ciliated cells produce secretory granules containing acid mucopolysaccharides. A discontinuous zonula occludens appears to be present.