Olfactory Sustentacular Cells Research Articles

Sustentacular Cell Enwrapment of Olfactory Receptor Neuronal Dendrites: An Update

The pseudostratified olfactory epithelium (OE) may histologically appear relatively simple, but the cytological relations among its cell types, especially those between olfactory receptor neurons (ORNs) and olfactory sustentacular cells (OSCs), prove more complex and variable than previously believed. Adding to the complexity is the short lifespan, persistent neurogenesis, and continuous rewiring of the ORNs. Contrary to the common belief that ORN dendrites are mostly positioned between OSCs, recent findings indicate a sustentacular cell enwrapped configuration for a majority of mature ORN dendrites at the superficial layer of the OE. After vertically sprouting out from the borderlines between OSCs, most of the immature ORN dendrites undergo a process of sideways migration and terminal maturation to become completely invaginated into and enwrapped by OSCs. Trailing the course of the dendritic sideways migration is the mesodendrite (mesentery of the enwrapped dendrite) made of closely apposed, cell junction connected plasma membrane layers of neighboring folds of the host sustentacular cell. Only a minority of the mature ORN dendrites at the OE apical surface are found at the borderlines between OSCs (unwrapped). Below I give a brief update on the cytoarchitectonic relations between the ORNs and OSCs of the OE. Emphasis is placed on the enwrapment of ORN dendrites by OSCs, on the sideways migration of immature ORN dendrites after emerging from the OE surface, and on the terminal maturation of the ORNs. Functional implications of ORN dendrite enwrapment and a comparison with myelination or Remak’s bundling of axons or axodendrites in the central and peripheral nervous system are also discussed.

Interferon gamma causes olfactory dysfunction without concomitant neuroepithelial damage

Olfactory loss is a debilitating symptom of chronic rhinosinusitis (CRS). The pathophysiology of inflammatory olfactory dysfunction likely involves both conductive and sensorineural components. To study the interaction of CRS-associated inflammatory cytokines with the olfactory epithelium (OE), a transgenic mouse model was developed that allows temporally-controlled local gene expression. Interferon-gamma (IFN-γ) is a prototypical T helper 1 (Th1) cytokine linked to nonpolypoid CRS (CRSsNP), as well as sinonasal viral and bacterial infections. In this study, the effects of chronic IFN-γ expression on olfactory histology and function were investigated. IFN-γ secretion by olfactory sustentacular cells was induced in the transgenic mouse. Viability and gross behavior were unaffected. Mice were euthanized after 6 weeks of IFN-γ expression, and olfactory tissue was studied by histology, immunohistochemistry, and electro-olfactography (EOG). Findings were compared with uninduced littermates. IFN-γ expression did not result in alteration of the normal histologic architecture of the neuroepithelium or lamina propria. However, EOG recordings demonstrated a significant decrease in odorant responses after IFN-γ expression. In addition, a marked increase in submucosal CD45-positive cells was observed, the majority of which were CD3-positive and CD4-positive lymphocytes. Chronic IFN-γ expression in the mouse OE results in diminished odorant responsiveness, despite the absence of inflammatory tissue damage. This suggests a direct effect of IFN-γ on olfactory neuron function that may underlie olfactory loss in CRSsNP or viral infections. The infiltration of submucosal lymphocytes raises the possibility that other downstream cytokines also contribute to olfactory dysfunction.

Loss of the V-ATPase B1 subunit isoform expressed in non-neuronal cells of the mouse olfactory epithelium impairs olfactory function.

The vacuolar proton-pumping ATPase (V-ATPase) is the main mediator of intracellular organelle acidification and also regulates transmembrane proton (H+) secretion, which is necessary for an array of physiological functions fulfilled by organs such as the kidney, male reproductive tract, lung, bone, and ear. In this study we characterize expression of the V-ATPase in the main olfactory epithelium of the mouse, as well as a functional role for the V-ATPase in odor detection. We report that the V-ATPase localizes to the apical membrane microvilli of olfactory sustentacular cells and to the basolateral membrane of microvillar cells. Plasma membrane V-ATPases containing the B1 subunit isoform are not detected in olfactory sensory neurons or in the olfactory bulb. This precise localization of expression affords the opportunity to ascertain the functional relevance of V-ATPase expression upon innate, odor-evoked behaviors in B1-deficient mice. This animal model exhibits diminished innate avoidance behavior (revealed as a decrease in freezing time and an increase in the number of sniffs in the presence of trimethyl-thiazoline) and diminished innate appetitive behavior (a decrease in time spent investigating the urine of the opposite sex). We conclude that V-ATPase-mediated H+ secretion in the olfactory epithelium is required for optimal olfactory function.

Olfactory epithelia exhibit progressive functional and morphological defects in CF mice

In normal nasal epithelium, the olfactory receptor neurons (ORNs) are continuously replaced through the differentiation of progenitor cells. The olfactory epithelium (OE) of the cystic fibrosis (CF) mouse appears normal at birth, yet by 6 mo of age, a marked dysmorphology of sustentacular cells and a dramatic reduction in olfactory receptor neurons are evident. Electroolfactograms revealed that the odor-evoked response in 30-day-old CF mice was reduced approximately 45%; in older CF mice, a approximately 70% reduction was observed compared with the wild type (WT) response. Consistent with studies of CF airway epithelia, Ussing chamber studies of OE isolated from CF mice showed a lack of forskolin-stimulated Cl(-) secretion and an approximately 12-fold increase in amiloride-sensitive sodium absorption compared with WT mice. We hypothesize that the marked hyperabsorption of Na(+), most likely by olfactory sustentacular cells, leads to desiccation of the surface layer in which the sensory cilia reside, followed by degeneration of the ORNs. The CF mouse thus provides a novel model to examine the mechanisms of disease-associated loss of olfactory function.

Ultrastructural Changes and Olfactory Deficits During 3-Methylindole-Induced Olfactory Mucosal Necrosis and Repair in Mice

Olfactory mucosa from C57BL/6N mice was examined by transmission electron microscopy at 0.5, 1, 2, 4, 6, 24, 48, and 72 h after 400 mg 3-methylindole (3MI)/kg ip, and at 7, 14, and 21 days after 300 mg 3MI/kg or vehicle. Degeneration was evident in epithelial cells of Bowman's glands and olfactory sustentacular cells by 0.5 h, but not in neurons until 24 h, by which time necrosis was fully developed in sustentacular cells and epithelium of Bowman's glands. Sustentacular cells and neurons detached from the basal lamina by 48 h. Lamina proprial fibroblasts were hypertrophied by 72 h and, with collagen fibrils, formed the bulk of the mucosa at 7days. By 21 days, fibroblasts were less conspicuous, but Bowman's glands were rarely observed and were lined by epithelial cells withoutsecretory granules. Mucosal epithelium was reconstituted, but disorderly and lacked olfactory differentiation. Mice treated with 3MI were more likely than control mice to taste water treated with isoamyl acetate (odorant) and quinine monohydrochloride (aversive tastant). Though olfactory neurons were initially spared, their absence in the regenerated epithelium explains lingering olfactory deficits in murine 3MI toxicosis.

The structure of the nasal chemosensory system in squamate reptiles. 1. The olfactory organ, with special reference to olfaction in geckos

The luminal surface of the chemosensory epithelia of the main olfactory organ of terrestrial vertebrates is covered by a layer of fluid. The source of this fluid layer varies among vertebrates. Little is known regarding the relative development of the sources of fluid (sustentacular cells and Bowman's glands) in reptiles, especially in gekkotan lizards (despite recent assertions of olfactory speciality). This study examined the extent and morphology of the main olfactory organ in several Australian squamate reptiles, including three species of gekkotans, two species of skinks and one snake species. The olfactory mucosa of two gekkotan species (Christinus marmoratus and Strophurus intermedius) is spread over a large area of the nasal cavity. Additionally, the sustentacular cells of all three gekkotan species contained a comparatively reduced number of secretory granules, in relation to the skinks or snake examined. These observations imply that the gekkotan olfactory system may function differently from that of either skinks or snakes. Similar variation in secretory granule abundance was previously noted between mammalian and non-mammalian olfactory sustentacular cells. The observations in gekkotans suggests that the secretory capacity of the non-mammalian olfactory sustentacular cells show far more variation than initially thought.

On the nature of rat hepatic and mouse olfactory sulfotransferases

Rat hydroxysteroid sulfotransferase (HS-SULT) cDNAs, ST-40 and ST-20 are 90% identical in amino acid sequences and show different substrate specificities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). ST-40 enzyme is active toward the three substrates, whereas ST-20 enzyme is preferentially active for CS. First we prepared mutants of well conserved histidine, lysine and asparagine by site-directed mutagenesis. Secondly we constructed 20 chimeric HS-SULTs by reciprocal exchange of five protein domains between ST-20 and ST-40 enzymes. The studies on the expressed mutant and chimeric enzymes indicate the importance of the C-terminal region for the substrate specificity and the involvement of multiple regions for the enzyme activities. Next we determined the genetic loci of ST-40 and ST-20 by fluorescence in situ hybridization. Biotinylated ST-20 and ST-40 probes gave a pair of fluorescent spots on the same region of rat chromosome 1 and the loci of these genes were localized to the same chromosomal region of 1q21.3→q22.1. Finally we studied mouse olfactory phenol SULT (P-SULT). It was immunolocalized in the cytoplasm of mouse olfactory sustentacular cells and mouse nasal cytosols show high SULT activities toward phenolic aromatic odorants. We subsequently isolated a mouse P-SULT cDNA from mouse olfactory cDNA library. It encodes 304 amino acid polypeptide and is 94% identical with rat ST1C1 in amino acid sequences.

Nasal Cytochrome P450 2A: Identification, Regional Localization, and Metabolic Activity toward Hexamethylphosphoramide, a Known Nasal Carcinogen

Two members of the cytochrome P450 2A subfamily, CYP2A10 and 2A11, are abundant nasal enzymes previously characterized in rabbit olfactory microsomes. Rabbit CYP2A is active toward a number of nasal toxicants, including the rat nasal procarcinogen hexamethylphosphoramide (HMPA). While P450s immunochemically related to the rabbit CYP2As have been detected in rat and human nasal mucosa, confirmation of these enzymes as members of the CYP2A subfamily and efforts to characterize their ability to bioactivate toxicants have been limited. In the present study, the regional distribution and cell-specific expression of CYP2A in the rat nasal cavity were examined using an antibody to rabbit CYP2A10/11. In sections of the anterior nose, immunoreactive CYP2A was present in ciliated cells of the nasal respiratory epithelium and cuboidal epithelial cells of the nasal transitional epithelium, but was absent in squamous epithelial cells. The most intense immunostaining was observed in the posterior nose. Olfactory sustentacular cells and Bowman's gland cells in sections posterior to the nasal papilla stained most intensely. Western blot analysis revealed that anti-CYP2A10/11 recognized a sharp band of approximately 50 kDa in nasal respiratory and olfactory microsomes, supporting the premise that the antibody is reacting with a cytochrome P450 enzyme. The nasal expression of CYP2A6 mRNA—a member of the human CYP2A subfamily having a high degree of homology to rabbit 2A10 and 2A11—was examined in human surgical patients. Middle turbinectomy tissues—largely composed of nasal respiratory epithelia—from 11 patients were analyzed for the presence of CYP2A6 using reverse transcription–polymerase chain reaction (RT–PCR). Identification of CYP2A6 was confirmed by DNA sequencing of RT–PCR products. CYP2A6 mRNA was detected in all of the human samples analyzed. In additional experiments, human CYP2A6 metabolized HMPA to formaldehyde, suggesting that this compound might cause nasal toxicity in humans. The identification of CYP2A cytochromes in rat and human nasal tissues may have important implications for risk assessment of inhaled xenobiotics.

Multiplicity of sulfotransferases

Sulfation is an important conjugation reaction in the metabolism of a diversity of xenobiotics and endogenous compounds such as drugs, food additives, carcinogens, steroids and neurotransmitters. Sulfate conjugation is catalyzed by various kinds of sulfotransferase (ST) such as hydroxysteroid ST (HS-ST), phenol ST (P-ST) and estrogen ST (E-ST) present in cytosols. Our laboratory has been studying the multiplicity of rat and mouse STs. We found that tertiary amines such as triethylamine selectively inhibited rat hepatic HS-ST. Developmental changes and zonal distributions of rat liver HS-ST and P-ST isoenzymes provided evidence for their complex regulatory mechanisms. Studies on site-directed mutagenesis and chimeras of rat HS-ST cDNAs, ST-40 and ST-20, revealed the importance of the C-terminal region for the substrate specificity and involvement of multiple regions for the enzyme activities. These two cDNAs have been mapped to the same chromosomal region 1q21.3-->q22.1 by fluorescence in situ hybridization. Mouse olfactory P-ST was present in the cytoplasm of olfactory sustentacular cells and its cloning study revealed that it is 94% identical with rat ST1C1 in amino acid sequences.

Zonal distribution of sulfotransferase for phenol in olfactory sustentacular cells.

We have immunolocalized phenol sulfotransferase (PST)G, an isoform of PST in sustentacular cells which reside in the dorso-medial portion of the nasal cavity of the mouse. The same topographical pattern of gene expression has been reported for some olfactory neuron-specific genes. When several established (phenol-containing) odorants were used as substrates, mouse nasal tissue cytosol showed a significant level of PST activity, as does mouse liver cytosol. This study is the first to demonstrate that gene expression in the olfactory sustentacular cells is also organized zonally, and indicates the involvement of sulfo-conjugation in olfactory perireceptor processes, such as odorant clearance and xenobiotic detoxification.

Cytoarchitecture of cultured olfactory neuroepithelial spheres: The role of olfactory sustentacular cells in cavity formation

When newborn rat olfactory mucosal cells were dissaggregated and plated as single cells onto astrocyte bed layers, abundant generation and maturation of olfactory receptor neurons was observed. Both immature and mature neurons were found in large multicellular spheres after 15 days in culture,. In sections of the spheres, almost all had central cavities, and the neurons were located almost exclusively in the external 1-2 cell layers. We show here that the spheres also contain olfactory sustentacular cells, neuronal supporting cells similar to retinal Muller cells. Sustentacular cell presence was shown by immunostaining cultured cells with a monoclonal antibody, SUS, which has previously been shown to recognize only sustentacular cells and a subset of the Bowman's gland cells. Fig. 1 shows that cultured cellular spheres contained numerous SUSpositive, roughly bipolar cells. These cells appear to be located internally rather than on the external surface. Sustentacular cell presence was further confirmed by immunostaining with a monoclonal antibody, 1F4, developed in this laboratory.

Monoclonal antibodies to the rat olfactory sustentacular cell

Immunization of BALB/c mice with rat olfactory epithelium permitted the production of a number of monoclonal antibodies directed towards cell types within the tissue. One of these antibodies, designated SUS-1, reacts specifically with cells located at the luminal surface of the olfactory neuroepithelium which possess a characteristic wine-glass body and a basal process which projects to the basement membrane. Thus these cells have the topographical and morphological properties of olfactory sustentacular cells. The SUS-1-positive cells are not stained by an antibody to the olfactory marker protein and neither does the SUS-1 antibody react with mature olfactory neurons in double labeling experiments. Upon unilateral olfactory bulbectomy there is a marked reduction in olfactory marker protein positive cells while there is no apparent diminution in the labeling of SUS-1 cells. These data show conclusively that the mature olfactory neurons and the SUS-1-positive cells are distinct from one another. SUS-1 staining is absent in all tissues examined except the olfactory epithelium. However, small discrete groups of cells in the lamina propria mucosae (often referred to as the submucosa) do stain in addition to the luminal sustentacular cell layer. The possibility is considered that the mature sustentacular cells may be related to these latter cells.

Pathology of toxic responses to the RD50 concentration of chlorine gas in the nasal passages of rats and mice

Male Swiss-Webster mice and Fischer-344 rats were exposed to chlorine gas at their respective RD50 concentrations (ca. 9 to 11 ppm). The RD50 concentration is that concentration which reduces respiratory rate by 50%. The exposures were carried out for 6 hr per day for 1, 3, or 5 days, and the animals were killed immediately at the end of the last exposure. The nasal passages were examined by light and scanning electron microscopy. Lesions were observed in all exposed groups and were of similar severity and character in rats and mice. The most severe changes were found in the olfactory mucosa of the anterior portion of the dorsal meatus and consisted of partial to complete degeneration of olfactory sensory cells, with olfactory sustentacular cells being more resistant to chlorine exposure. Lesions in the respiratory epithelium were located primarily on the free margins of the naso- and maxilloturbinates and adjacent nasal septum. Scanning electron microscopy, using large size specimens, demonstrated loss of olfactory cilia in areas of the olfactory epithelium which appeared unaffected by light microscopy. Scanning electron microscopy was also helpful in locating areas of respiratory epithelium exhibiting loss of cilia and cellular exfoliation, which occurred primarily on naso- and maxilloturbinates. Therefore, chlorine-induced severe lesions in specific locations in both the olfactory and respiratory epithelia of the nasal passages with more widespread loss of respiratory and olfactory cilia.