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.     

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.     

''Ciliated'' Tumour Cells In Ascitic Fluid From Two Cases of Cystadenocarcinoma of the Ovary

We report two cases of primary carcinoma of the ovary in which 'ciliated' adenocarcinoma cells were found in the ascitic fluid. Transmission electron microscopy revealed that these were not true cilia but rather a prolific growth of abnormal microvilli. The cytological findings were compared with the histological appearances of the primary tumour. No ciliated cells were seen in the primary tumour, suggesting that the formation of the microvilli represented an independent proliferation of the cells in the fluid. Special staining reactions for mucin, alkaline phosphatase and epithelial membrane antigen were identical in the primary tumour and the cells in the ascitic fluid.     

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     

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.      

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.     

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.     

Surface specializations of the olfactory epithelium of rainbow trout, Salmo gairdneri

Receptors for olfactory stimulus molecules appear to be located at the surface of olfactory receptor cells. The ultrastructure of the distal region of rainbow trout (Salmo gairdneri) olfactory epithelium was examined by transmission electron microscopy. On the sensory olfactory epithelium, which occurs in the depressions of secondary folds of the lamellae of the rosettes, five cell types were present. Type I cells have a knob-like apical projection which is unique in this species because it frequently contains cilia axonemes within its cytoplasm in addition to being surrounded by cilia. Type II cells bear many cilia oriented unidirectionally on a wide, flat surface. Type III cells have microvilli on a constricted apical surface and centrioles in the subapical cytoplasm. Type IV cells contain a rod-like apical projection filled with a bundle of filaments, and type V cells are supporting cells. Cilia on the sensory epithelium contain the 9 + 2 microtubule fiber pattern. Dynein arms are clearly present on the outer doublet fibers, which suggests that the cilia in the olfactory region are motile. Their presence in olfactory cilia of vertebrates has been controversial. The cilia membrane in this species is unusual in often showing outfoldings, within which are included small, irregular vesicles or channels. In addition, cilia on type II cells frequently contain dense-staining bodies closely apposed to the membranes, along with a densely stained crown at the cilia tip. Previous biochemical evidence indicates that odorant receptors are associated with the cilia.     

Vomeronasal receptors in Turtles

Summary The functional similarities observed with electrophysiological techniques between olfactory and vomeronasal receptors allow speculation that morphological details essential to the common function should be observed in both cases. Both mucosae have primary receptors within the epithelium which are surrounded, but not completely isolated, by so-called supporting cells. These last secrete a granular product. In both epithelia receptor cells contact each other at the axonal, perikaryal, dendritic and junctional complex levels. The axons of the two types of receptors are unmyelinated and their diameter ranges from 0.1 to 0.4 micron. The most interesting difference between the two types of receptors lies at the level of their exposed endings. The olfactory vesicle, as it is classically represented in olfactory receptors and is common in those of turtles in the form of a ball-like protrusion above the epithelial surface, is usually missing in the vomeronasal receptors. These have a tapering cone-shaped irregular projection always complicated by a set of branched microvilli. They do, furthermore, consistently lack cilia. This observation is in agreement with recent TEM observations. The assumption that cilia are essential in the mechanism of olfactory transduction is discussed on the basis of these anatomical findings.     

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.     

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).     

The distribution of dopamine-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria)

Summary An indirect gold-labeling method utilizing the lectin from Limax flavus was employed to characterize the subcellular distribution of sialic acid in glycoconjugages of the salamander olfactory mucosa. The highest density of lectin binding sites was in secretory vesicles of sustentacular cells. Significantly lower densities of lectin binding sites were found in secretory granules of acinar cells of both Bowman's and respiratory glands. Lectin binding in acinar cells of Bowman's glands was confined primarily to electron-lucent regions and membranes of secretory granules. In the olfactory mucus, the density of lectin binding sites was greater in the region of mucus closest to the nasal cavity than in that closest to the epithelial surface. At the epithelial surface, the density of lectin binding sites associated with olfactory cilia was 2.4-fold greater than that associated with microvilli of sustentacular cells or non-ciliary plasma membranes of olfactory receptor neurons, and 7.9-fold greater than non-microvillar sustentacular cell plasma membranes. Lectin binding sites were primarily associated with the glycocalyx of olfactory receptor cilia. The cilia on cells in the respiratory epithelium contained few lectin binding sites. Thus, sialylated glycoconjugates secreted by sustentacular cells are preferentially localized in the glycocalyx of the cilia of olfactory receptor neurons.     

The ultrastructural organization and the alkaline phosphatase activity of the epithelial surface of the bovine fallopian tube

Summary The epithelial surface of the bovine Fallopian tube has been studied with respect to its ultrastructure and alkaline phosphatase activity. The ciliated cells were found to be provided with cilia and microvilli. The cilia were placed in rows and were most frequent in the central area of each cell surface. The basal corpuscles were provided with indistinct rootlets. The microvilli occurred between the cilia, but showed the reverse distribution, being most frequent towards the periphery of the cell surface. As a rule a marked alkaline phosphatase activity could be revealed on the ciliated cell surface, presumably associated with the microvilli.The secretory cells were bulging over the surface of the ciliated cells. Theywere provided with irregular protrusions, which were as a rule long and slender during the follicular phase but shorter and bulkier during the luteal phase. No alkaline phosphatase activity was detected.This investigation was supported by a grant from the foundation Thérèse och Johan Anderssons Minne.     

Ultrastructural Aspects of Olfactory Signaling

The olfactory area of the nasal cavity is lined with olfactoryreceptor cell cilia that come in contract with incoming odormolecules. Ultrastructural immunocytochemical studies in rodentshave shown that these cilia contain all the proteins necessaryto transduce the odorous message into an electrical signal thatcan be transmitted to the brain. These signaling proteins includeputative odor receptors, GTP binding proteins, type III adenylylcyclase and cyclic nucleotide-gated channels. The rest of thecells, including dendrites and dendritic knobs, showed no discerniblelabeling with antibodies to these signaling proteins. Furthermore,freeze-fracture and freeze-etch studies have shown that themembrane morphology of olfactory cilia differs substantiallyfrom that of non-sensory cilia. Olfactory cilia have many moremembrane particles. Transmembrane signaling proteins, such asodor receptors, adenylyl cyclase and cyclic nucleotide-gatedchannels, conceivably appear as membrane particles. Thus, thelong-standing supposition that olfactory cilia are peculiarlyadapted to deal with the reception and initial transductionof odorous messages has now been verified in terms of both ultrastructuralmorphology and cytochemistry. Emerging studies on vomeronasalreceptor cell microvilli indicate that the same is true forthis organ, even though the actual signaling components differfrom those of the main olfactory system. Chem. Senses 22: 295–311,1997.     

Ultrastructure of the nuchal organ in the interstitial polychaete Stygocapitella subterranea (Parergodrilidae)

The nuchal organs of Stygocapitella subterranea are paired narrow pits. They are lined by unciliated cells at the opening and by ciliated cells at the basal parts. The primary sensory cells (6–8) are arranged in a single patch at the bottom of the nuchal pit. The nuclei of the sensory cells are located in the posterior portion of the brain. Their dendrites form the nuchal nerve which is sheathed by the ciliated cells. Each sensory cell bears up to 4 modified sensory cilia and several microvilli extending into the olfactory chamber. The sensory cilia show various patterns of axonemal organization and have no rootlets. The olfactory chamber is covered by a cuticular matrix. Another primary sensory cell lies at the opening of the nuchal pit. It bears cilia which penetrate the cuticle but are enveloped by the epicuticle. Retractor muscles insert caudally on the organ. The nuchal organ of S. subterranea shows similarities to those of opheliids but exhibits several features not to be found in other nuchal organs.     

Identification of a group of novel membrane proteins unique to chemosensory cilia of olfactory receptor cells

We have used a library of monoclonal antibodies (mAbs) against chemosensory cilia of the olfactory epithelium of Rana catesbeiana to identify proteins that are unique to the ciliary membrane. Five different antibodies (mAb 8, 26, 34, 42/45, and 43) identify novel proteins in olfactory cilia that are not detected in olfactory nerve membranes, nonchemosensory cilia from respiratory epithelium, or membranes from brain, heart, liver, kidney, and lung. Deglycosylation of olfactory cilia with endoglycosidase H shows that most of these antibodies (mAb 8, 42/45, 43, and possibly 26) react with antigenic determinants comprised partially or entirely of carbohydrate, while only one (mAb 34) recognizes an 87-kDa protein that is resistant to endoglycosidase H treatment. Furthermore, a 59-kDa glycoprotein visualized by mAb 8 exists as membrane-associated oligomers connected via intermolecular disulfide bonds. These proteins, tagged with distinct high-mannose-containing carbohydrate moieties and found only in chemosensory cilia of olfactory receptor cells, may be involved in odorant recognition and/or olfactory transduction.     

The ultrastructural organization of olfactory epithelium of two species of gadoid fish

The untrastructural organization of the olfactory epithelium of the cod Gadus morhua (L.) and the haddock Melanogrammus aeglefinus (L.) was studied using both transmission and scanning electron microscopy. The olfactory rosette was found to exhibit regional differences; the faces of the olfactory lamella were composed of sensory epithelium, the edges were non-sensory. The cellular organization of the olfactory epithelium was determined and consisted of bi-polar sensory neurones, supporting cells, mucous cells and basal cells. The ultrastructure of the sensory cells was consistent, having an elongate cell, the free surface of which terminated in an olfactory vesicle from which arose either four olfactory cilia or numerous microvilli. Ciliary aggregations have been found in the two species of gadoid fish studied; it is suggested that these structures aid in the separation and in the circulation of fluid between the lamellae. The surface structure of the supporting cells was found to be of two types: either ciliated or ridged; the former presenting distinct ciliated tufts, the latter showing definite, but unorganized, ridges over the epithelium surface.     

Cytochemical localization of adenylate cyclase activity in rat olfactory cells

Summary Adenylate cyclase activity was demonstrated in the cilia, dendritic knob and axon of rat olfactory cells by using a strontium-based cytochemical method. The activity in the cilia and the dendritic knob was enhanced by non-hydrolyzable GTP (guanosine triphosphate) analogues and forskolin, and inhibited by Ca²⁺, all in agreement with biochemical reports of the odorant-sensitive adenylate cyclase. The results support the hypothesis of cyclic AMP working as a second messenger in olfactory transduction and imply that the transduction sites exist not only in the olfactory cilia but also in the dendritic knob. Enzymatic activity was also observed in the olfactory dendritic shaft by treating the tissue with 0.0002% Triton X-100, although the properties and role of the enzyme in this region are uncertain. The detergent inhibited the enzymatic activity in the cilia and the dendritic knob.     

Receptor cells of different types and quantitative relations between them in the organ of smell of larval and adult specimens of acipenserid fishes]

Three types of olfactory cells: rod-like and cone-like (flagellar olfactory cells) and filamentous (microvillar olfactory cells), which have been described previously in adult Acipenseridae were found in the olfactory organ of the ten-days larval sturgeons (Acipenser güldenst?dti), sevrugas (Acipenser stellatus) and sterlets (Acipenser ruthenus). The flagellar olfactory receptors appeared to predominate in both ten-days larvae and adults of the anadromous sturgeons and sevrugas, while the microvillar olfactory receptors predominate in freshwater sterlets in ten-days larvae as well in adults. The facts obtained confirm the idea that the rod-like, cone-like and filamentous olfactory cells are independent types of olfactory receptors. The different ratios of these cells in the olfactory organs of anadromous and fresh-water Acipenseridae may be a result of their ecological adaptations.     

Electron-microscopic demonstration of olfactory-marker protein with protein G-gold in freeze-substituted,Lowicryl K11M-embedded rat olfactory-receptor cells

Summary In this study electron-microscopic immunocytochemistry was used to localize olfactory marker protein in olfactory epithelia. Rat olfactory-epithelial samples were rapidly frozen, freeze-substituted with acetone, embedded at low temperatures with Lowicryl K11M and labelled on the sections with polyclonal antibodies raised against olfactory marker protein and with protein G conjugated to colloidal gold. Apart from the aforementioned use of acetone, substitution was carried out in the complete absence of chemical fixation, i.e., neither aldehydes nor OsO⁴ were used. This procedure resulted in localization concurrent with a good ultrastructural preservation. Olfactory-marker protein was present throughout the cytoplasmic compartments of dendrites and dendritic endings of olfactory-receptor cells, but it was not found in organelles such as mitochondria. Olfactory-marker protein was found only in dendriticendings of olfactory-receptor cells mature enough to have given rise to cilia, but these cilia displayed less labelling than dendrites and dendritic endings. Olfactory-marker protein was not found in apices and microvilli of neighboring olfactory-supporting cells.