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).     

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     

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.     

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.     

A morphometric comparison of the olfactory epithelium of newborn and weanling rabbits

Summary As part of a study of the development of olfactory function in the rabbit, a morphometric analysis of the olfactory epithelium in newborn and 30-day-old animals was carried out. Surface area, thickness and cell densities of the olfactory epithelium were compared in hematoxylin-eosin stained serial sections through the nasal cavities of 4 newborn and 3 weanling rabbits. While the basic structure of the olfactory cavity changed little with age, a large quantitative development in the epithelium was observed. The pattern of growth appeared uniform and resulted in a 3-fold increase in total surface area from about 1 cm² per side in the newborn to about 3 cm² in the weanling, and an increase in thickness from approximately 65 m to about 90 m. The increase in thickness was due mainly to a disproportionate, 5-fold increase in the number of olfactory neurons. This resulted in a total of about 32 million cells per side by day 30, and represented an increase in the ratio of neurons to basal cells of 7:1 to 10:1, and neurons to supporting cells of 2:1 to 4:1. While such an increase in the number of primary neurons presumably improves the animal's perceptual abilities, it nevertheless raises the question as to how perceptual constancy can be maintained during a period of such rapid neural change.     

Replacement of receptor cells in the hamster vomeronasal epithelium after nerve transection

Chemoreceptor cells in the vomeronasal and olfactory epithelium are replaced following experimentally induced degeneration. This study analyzes quantitatively the time course and degree of vomeronasal receptor cell replacement. Unilateral transection of the vomeronasal nerves in adult hamster was used to induce a retrograde degeneration of receptor cells in the vomeronasal organ. Histological measurement of both number of receptor cells and epithelial thickness were made for recovery times from 0 to 60 days. After nerve transection, there was a gradual degeneration of receptor cells, the number decreasing to 50% of control by day 2 and 16% by day 6. During days 7-15 maximum receptor cell replacement was observed. Cell number increased rapidly and reached a peak on day 15. At recovery times of 40-60 days, cell number returned to the control level. Epithelial thickness, however, decreased to 60-70% during the degeneration period (days 4-6) and did not return to control levels. After 40-60 days epithelial thickness remained at 70% of control. These results demonstrate that vomeronasal receptor cells are replaced following degeneration, but epithelial thickness does not return to control levels. These findings suggest that the number of replacement cells is not limited by the reduced thickness of the epithelium, and that recovery mechanisms may function to restore an optimum number of receptor cells.      

Olfactory epithelium consisting of supporting cells and horizontal basal cells in the posterior nasal cavity of mice

The olfactory epithelium of mice generally consists of olfactory cells, progenitors of olfactory cells (globose basal cells), supporting cells, and horizontal basal cells. However, in the dorsal fossa (the roof) of the posterior nasal cavity of mice, we found seven epithelial patches consisting of only non-neuronal cell types, i.e., supporting cells and horizontal basal cells, among the normal olfactory epithelium. The supporting cells occupied three or four layers in the apical to middle regions; in the basal region, horizontal basal cells were localized in a single row adjacent to the basement membrane. Bowman's gland ducts were also present in the epithelium. Neuronal cells (olfactory cells and globose basal cells) were totally absent. The ultrastructure of the supporting cells, horizontal basal cells, and Bowman's glands was essentially similar to that in the normal olfactory epithelium. In the early postnatal period (P1-P7), cell types in the epithelium were the same as those in the normal olfactory epithelium. From P10 to P21, olfactory cells and globose basal cells had disappeared from the olfactory epithelium. At this period, the number of TUNEL-positive cells was significantly higher than that in the surrounding olfactory epithelium; ultrastructurally, many apoptotic figures were observed. This suggests that the epithelium consisting of supporting cells and horizontal basal cells is generated by the apoptotic death of olfactory cells and globose basal cells during postnatal development.     

The cell coat of the sensory and supporting cells of the rainbow trout saccular macula as demonstrated by reaction with ruthenium red and tannic acid.

The surface of most cells is covered by glycoconjugates. The composition and thickness of the surface coat varies among different cell types. The purpose of the present study was to demonstrate the presence of and to characterize the cell coat surrounding the cells in the saccular macula of the rainbow trout. Tissues were fixed in Karnovsky's fixative containing either ruthenium red (0.5, 1, or 2%) or tannic acid (1, 2, or 4%). The apical surface of the sensory and supporting cells reacted with both agents. Varying the concentration of the compounds within a certain range did not significantly affect the degree of tissue staining. Whereas ruthenium red staining was distributed evenly along the luminal surface of the epithelium and along the length of the stereocilia, tannic acid formed electron-dense clumps on the luminal surface of sensory and non-sensory cells and in the basal region of the macular epithelium. The stereocilia of the sensory cells also exhibited tannic acid-positive, electrondense precipitate, particularly near the distal ends of these processes, while uniform staining of the plasma membrane was seen along their lengths. The results of this study suggest that the trout saccular macula is provided with extracellular microenvironments which may be necessary for functional integrity.     

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.     

Olfactory mucosa ultrastructure in the short-tailed shrews,Blarina brevicauda and Blarina carolinensis

The olfactory mucosae of the northern short-tailed shrew, Blarina brevicauda, and the southern short-tailed shrew, Blarina carolinensis, were examined by light and electron microscopy. A well-developed olfactory epithelium was observed that included all of the cells necessary to provide for a sensitive olfactory system, suggesting that olfaction plays a major role in the behavior of these animals. There were no significant differences between the olfactory mucosae of these two species. The general features of the olfactory epithelium in these shrews were similar to those reported for several other macrosmatic mammals. A new type of supporting cell, called the light supporting cell, was observed in these shrews. The light supporting cell cytoplasm exhibited very little staining by light microscopy and had low electron density by transmission electron microscopy compared to that of the more common dark supporting cell. The light supporting cell had a convex apical surface with microvilli and lacked the large amounts of smooth endoplasmic reticulum (SER) typical of the apical cytoplasm of the dark supporting cell. In the lamina propria of the mucosa, the Bowman's glands consisted of two cell types, one with electron-lucent, alcian blue-positive granules, and the other with electron-dense PAS-positive granules. The cell with electron-lucent granules contained large amounts of SER and small clumps of rough ER. The cells with electron-dense granules had large amounts of RER and little SER.     

Effects of methyl bromide on the rat olfactory system

The nature and extent of damage produced by methyl bromide (MeBr)exposure, and recovery of function after exposure, were studiedusing a multifacct approach which included behavioral, morphologicaland neurochemical endpoints. Thirty adult male Long–Evansrats were exposed to 200 p.p.m. MeBr for 4 h/day, 4 days a weekfor 2 weeks. Fifteen control rats were exposed to filtered aironly. On the first day following the onset exposure to MeBr,extensive damage to the olfactory epithelium as well as greatlyimpaired olfactory function were observed. However, even withcontinuous MeBr exposure, olfactory function was essentiallynormal after 4 days of exposure. Repair of the epithelium wasin progress by day 4 although morphology was atypical. The levelsof carnosine in both the olfactory epithelium and bulbs wereseverely depleted by day 4. Recovery, both in terms of structuralrepair and return of normal carnosine concentrations, laggedfar behind recovery of the ability to detect an odor stimulus.Even with repeated exposure, olfactory function recovered rapidly,even faster than anatomical repair. Measurement of overall carnosinelevels correlated well with the results obtained from representativeareas of tissues selected for histopathology. Morphometric analysisprovided quantitative detail on the nature of insult resultingfrom MeBr exposure. These data indicate that the olfactory systemis a most resilient system and that normal function is possibleeven after repeated insult by a toxic agent.     

Effects of reversible nare occlusion on the development of the olfactory epithelium in the rabbit nasal septum

Summary To investigate environmental influences on the development of the olfactory epithelium, semi-thin sections were taken from the nasal septum of newborn and 30-dayold rabbits; the epithelial thickness and the number of olfactory knobs, supporting cells, dark basal cells, and receptor cells were compared. During normal development, a marked increase in epithelial thickness was found, largely because of an increase in the number of receptor cells. Whereas unilateral nare occlusion on day 1 resulted in 10% fewer receptor cells and 25% fewer knobs on the deprived side by day 30, nare occlusion either up to or after day 5 had little effect, and even temporary reopening from days 6–7 was sufficient to stimulate receptor-cell development on the occluded side. Although in these latter cases, a slight deprivation effect of 6% was still found in the number of receptor-cell nuclei, there was no longer a significant difference in the number of knobs between the open and closed sides. Thus, whereas exposure to the environment during the first days of life appears to be sufficient to stimulate sustained growth, the deprived epithelium may retain the capacity to respond to such cues beyond this time. However, as nare occlusion also had an effect on the respiratory epithelium and nasal lymphatic tissue, the nature of the cues stimulating receptor-cell development, whether olfactory or non-olfactory, is not yet clear.     

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.     

Time-course of apoptosis in the olfactory epithelium of rainbow trout exposed to a low copper level

Copper at low doses is known to specifically induce olfactory neuron death in fish olfactory epithelium. Using light and electron transmission microscopy, we have investigated the features and the time-course of receptor cell death in rainbow trout exposed for 15 days to 20 mug Cu(2+)/l. Ultrastructural observations demonstrate that degenerating cells, which included both mature and immature neurons, exhibited morphological changes characteristic of a cell death by apoptosis. Quantitative analysis shows that the number of apoptotic cells increased significantly already after 1 day of exposure, reaching a peak at day 5. From this timepoint of exposure, no more mature neuron was noted in the olfactory epithelium. Following a significant decrease in the number of apoptotic cells at day 10, a second wave of neuron death was noted at day 15. These findings argue for the occurrence of a neurogenesis process to balance the receptor cell death, despite continued copper exposure, and for a higher vulnerability to the metal of olfactory neurons presenting more advanced stages of cell differentiation. The molecular mechanisms by which copper may induce olfactory neuron apoptosis are discussed.     

Olfactory marker protein immunohistochemistry and the anterograde transport of horseradish peroxidase as indices of damage to the olfactory epithelium

The present study compared the relative effectiveness of wheatgerm agglutinin--horseradish peroxidase (WGA--HRP) and olfactory marker protein (OMP) in detecting the presence of intact olfactory axons in glomeruli of the main olfactory bulb (MOB) in the rat. The olfactory epithelium was damaged by i.p. injections of the toxin 3-methyl indole and, after 5 or 6 days, the olfactory sac was injected with a 1% WGA--HRP solution. No anterograde labeling was observed in the dorsal and ventromedial quadrants of the MOB in the WGA--HRP material. However, in the same cases OMP immunostaining was observed throughout the MOB. In other rats the rostral olfactory epithelium was aspirated unilaterally and after 3, 11 and 16 days the olfactory sacs were injected with WGA--HRP and rats were perfused 1 day later. In these cases WGA--HRP reaction product was absent in the dorsolateral quadrant of the MOB on the aspirated side in all animals, but OMP immunostaining could be detected in the 4 and 12 day survival animals but not in the 17 day survival rat. These findings indicate that anterograde transport of WGA--HRP accurately reflects the presence of intact axons en route to the MOB. In contrast, OMP immunostaining persists in axon terminals severed from their parent cell body for at least 12 days and is a less useful marker of intact olfactory axons in experiments using short survival times.     

The interphotoreceptor space. I. Postnatal ontogeny in mice and rats

The postnatal ontogeny of the retinal interphotoreceptor space of mice and rats was studied by electron microscopy to elucidate the cytological developments in the surrounding cells relevant to the accumulation of extracellular glycosaminoglycans and glycoproteins constituting the interphotoreceptor matrix. This extracellular material at birth is principally the cell coat on all the immature cells that border the space at that time, but later additional weblike strands are seen in the space. The cells delimiting the space in the adult are the pigment epithelium (PE), the photoreceptors, and the glial cells of Müller. The Golgi complex of the PE is large at birth but involutes by day 15. Melanogenesis is the principal activity in this cell at birth but as the melanin granules mature, lysosomes gradually accumulate. Growth of the apical microvilli of the PE is most pronounced between day 5 and 15, which is also the time of rapid expansion of the interphotoreceptor space. The Golgi complex of the photoreceptor enlarges during this interval also, and remains voluminous thereafter. Müller's cells insert only slender apical processes lacking in secretory vesicles, into the interphotoreceptor space. All the adult cells have a cell coat on the surfaces facing the interphotoreceptor space. Secretory vesicles were not identified in any of the cells impinging on the space.     

Morphogenesis of the antenna of the male silkmoth, Antheraea polyphemus. II. Differential mitoses of 'dark' precursor cells create the Anlagen of sensilla

The antenna of the male silkmoth Antheraea polyphemus develops from a one-layered, flattened epidermal sac during the pupal phase. Within the first day post-apolysis (developmental stages 1 and 2), this epithelium differentiates into 'sensillogenic' and 'nonsensillogenic' regions, while numerous slender 'dark cells' interpreted as the precursor cells of sensilla arise in the former. Approximately between the first and second day post-apolysis (developmental stage 3), the dark cells retract to the apical pole of the epidermis, assume a round shape, and undergo a series of differential mitoses with spindles usually oriented parallel to the epidermal surface. These mitoses finally yield the Anlagen of the olfactory sensilla trichodea, each consisting of mostly 6-7 dark cells arranged side by side. In most of the Anlagen, 3-4 of these cells are situated more basally, each giving off a slender apical process which together are arranged in a fascicle. These are the prospective 2-3 sensory neurons plus the thecogen cell, which most probably is a sister cell of the former. Three additional cells are arranged more apically and partly enclose the fascicle of presumed sensory and thecogen cell processes. These are interpreted as the trichogen plus 2 tormogen cells, one of the latter degenerating later during development. In the basal region of the sensillogenic epidermis, massive signs of cell degeneration have been found. At stage 3, the basal epidermal feet in the non-sensillogenic regions have assumed a more uniform orientation as compared with the preceding stages.     

Immunohistochemical detection of EGF and NGF receptors in human olfactory epithelium.

The immunohistochemical localization of EGF and NGF receptors has been studied in the olfactory epithelium of human foetuses from 8 to 12 weeks of age. A positivity for EGF receptor, increasing with the age, was detected in the apical portion of the sensory epithelium. The NGF receptor was well detectable also at 8 weeks and localized both in differentiated olfactory cells and in some basal cells. From primary cultures of olfactory epithelium, a cell clone positive for Enolase, Neurofilaments and S-100 Protein was identified. These cells were shown to be reactive for EGF and NGF receptors. The addition of Retinoic acid to the culture medium induces a morphological differentiation of these cells that become positive for the Olfactory Marker Protein.     

Transregulation of erbB expression in the mouse olfactory bulb.

Previously, we have shown that erbB-3 expression is restricted to the ensheathing cells of the olfactory nerve layer, while erbB-4 is found in the periglomerular and mitral/tufted cells of the olfactory bulb and in cells coming out from the rostral migratory stream of the subependymal layer. In the present work, we have treated adult mice with zinc sulfate intranasal irrigation and analyzed erbB-3 and erbB-4 expression in the deafferented olfactory bulb. Following treatment, olfactory axons undergo degeneration, as indicated by the loss of OMP expression in the deafferented olfactory bulb. The thickness of the olfactory nerve layer is reduced, but the specific intensity of erbB-3 labeling in the remaining olfactory nerve layer is increased with respect to control. Interestingly, following deafferentation, erbB-4 immunoreactivity decreases specifically in cell types that normally make synaptic contacts with primary olfactory neurons in the glomeruli, i.e. periglomerular and mitral/tufted cells. Partial lesion of the olfactory epithelium allows regenerative axon growth of olfactory neurons to the olfactory bulb. Following olfactory axon regeneration, erbB-3 and erbB-4 immunoreactivity in the olfactory bulb is similar to control. Thus, like tyrosine hydroxylase, the down regulation of erbB-4 expression in the periglomerular cells is reversible.     

Immunocytological studies on an estradiol sensitive antigen in the cervicovaginal epithelium of neonatal mice

Summary Cells of the cervicovaginal epithelium of neonatal mice underwent morphological changes in response to estradiol injection. On the luminal border, estradiol treatment caused development of distinct microvilli and a prominent surface coat of delicate filamentous material. Very deep nuclear folds appeared, and the border between adjacent cells became strongly interdigitated. The cells developed a pronounced smooth and rough endoplasmic reticulum, and dark-stained membrane-bounded granules accumulated in the apical part of the cells.Estradiol promoted increased production of an antigenic material specific for the cervicovaginal epithelium (CVA). Immunofluorescence studies demonstrated CVA in the most apical part of the cells, in the extracellular material on the epithelial surface, and in the intercellular spaces between adjacent epithelial cells. This was confirmed by immunoferritin methods, which revealed that the antigen was localized to the surface coat and to material adhering closely to the exterior of the cell membrane, the part facing the lumen and also the part facing intercellular spaces. Within the cells, ferritin tagging was recognized around the membranes enclosing the dark-stained granules in the apical part of the cells and also on the inside of the luminal cell membrane. This is so interpreted that CVA acquires its antigenic properties when passing out from the dark-stained granules through the surrounding envelope. CVA apparently forms part of the glycocalyx of the cervicovaginal cells.This investigation was supported by grants from the Norwegian Cancer Society (Landsforeningen mot Kreft) and the Norwegian Research Council.