Aquaporin pathways and mucin secretion of Bowman's glands might protect the olfactory mucosa

The sense of smell is conveyed by the olfactory sensory neurons of the olfactory mucosa. Uniquely for sensory systems, the olfactory neurons directly face the external environment and are thus vulnerable to infections and changes in the airway surface liquid, but the surface liquid production and maintenance is not well understood. Here we show in rats and mice that Bowman's glands secrete the mucin MUC5AC. Aquaporin-5 was present at the apical face of the olfactory epithelium, completing a water transport pathway to the surface of the epithelium. Immunogold electron microscopy analysis revealed an intricate network of fine Aquaporin-1-positive fibroblast processes that surround Bowman's glands, whereas deeper blood vessels were unlabeled for Aquaporin-1. Our results show how the olfactory mucosa might be protected against infections and dehydration generally and how neuronal function is protected against ion concentration changes in the airway surface liquid by rapid replacement of water losses through the aquaporin pathways.     

Distribution and severity of spontaneous lesions in the neuroepithelium and Bowman’s glands in mouse olfactory mucosa: age-related progression

Age-related changes were examined in the distribution and severity of spontaneous lesions in the neuroepithelium and Bowman’s glands in mouse olfactory mucosa. The olfactory mucosa of female ICR mice at postnatal ages from 10 days to 16 months were investigated histologically by hematoxylin and eosin staining, high-iron diamine-Alcian blue (HID-AB) staining, and immunohistochemistry for olfactory marker protein (OMP), βIII tubulin (βIIIT), and Ki67. The lesions in the neuroepithelium and Bowman’s glands were quantitatively assessed by morphometric analyses of sections stained with anti-OMP antibody or HID-AB. The first appearance of neuroepithelial abnormality was observed in the dorsomedial portion of the olfactory mucosa in 5-month-old mice. The distribution and severity of lesions progressed with increasing age. In mildly affected epithelium in which OMP-positive olfactory receptor neurons (ORNs) were present but in smaller amounts, the numbers of βIIIT-positive and Ki67-positive neuroepithelial cells tended to be increased, indicating that neurogenesis was upregulated in these areas. In contrast, severely affected epithelium in which OMP-positive ORNs were virtually absent showed high variability in the numbers of βIIIT- and Ki67-positive cells among the areas examined, probably reflecting differences in the capacity of the basal cells remaining in the affected area to generate new neuronal cells. Histological analysis with HID-AB revealed that spontaneous lesions in Bowman’s glands also occurred in aged mouse olfactory mucosa. Lesions in the neuroepithelium and underlying Bowman’s glands tended to be spatially co-localized, suggesting a close association between pathogeneses in these two structures. Moreover, lesions in Bowman’s glands were associated with changes in the biochemical composition of mucus on the olfactory mucosa. This information should prove useful in improving the understanding of the pathogenetic mechanisms underlying age-related changes in the peripheral olfactory system. This work was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (nos. 14770886, 16790987 and 18799002; K. Kondo) and a grant from the Japanese Ministry of Health, Labour, and Welfare (Comprehensive Research on Aging and Welfare, no. H13-choju-012; K. Nibu).     

Lack of fibulin-3 alters regenerative tissue responses in the primary olfactory pathway

The adult olfactory epithelium has maintained the ability to reconstitute its olfactory sensory neurons (OSNs) from a basal progenitor cell compartment. This allows for life-long turnover and replacement of receptor components as well as repair of the primary olfactory pathway in response to injury and environmental insults. The present study investigated whether fibulin-3, a glycoprotein in the extracellular matrix and binding partner of tissue inhibitor of metalloproteinases-3 (TIMP-3), plays a role in ongoing plasticity and regenerative events in the adult primary olfactory pathway. In wild-type control mice, fibulin-3 protein was detected on IB4⁺CD31⁺ blood vessels, nerve fascicles and the basement membrane underneath the olfactory epithelium. After target ablation (olfactory bulbectomy), fibulin-3 was also abundantly present in the central nervous system (CNS) scar tissue that occupied the bulbar cavity. Using two different lesion models, i.e. intranasal Triton X-100 lesion and olfactory bulbectomy, we show that fibulin-3 deficient (Efemp1^?/?) mice have impaired recovery of the olfactory epithelium after injury. Ten days post-injury, Efemp1^?/? mice showed altered basal stem/progenitor cell proliferation and increased overall numbers of mature (olfactory marker protein (OMP) -positive) versus immature OSNs. However, compromised regenerative capacity of the primary olfactory pathway in Efemp1^?/? mice was evidenced by reduced numbers of mature OSNs at the later time point of 42 days post-injury. In addition to these neural differences there were consistent changes in blood vessel structure in the olfactory lamina propria of Efemp1^?/? mice. Overall, these data suggest a role for fibulin-3 in tissue maintenance and regeneration in the adult olfactory pathway.     

Adiponectin Enhances the Responsiveness of the Olfactory System

The peptide hormone adiponectin is secreted by adipose tissue and the circulating concentration is reversely correlated with body fat mass; it is considered as starvation signal. The observation that mature sensory neurons of the main olfactory epithelium express the adiponectin receptor 1 has led to the concept that adiponectin may affect the responsiveness of the olfactory system. In fact, electroolfactogram recordings from olfactory epithelium incubated with exogenous adiponectin resulted in large amplitudes upon odor stimulation. To determine whether the responsiveness of the olfactory sensory neurons was enhanced, we have monitored the odorant-induced expression of the immediate early gene Egr1. It was found that in an olfactory epithelium incubated with nasally applied adiponectin the number of Egr1 positive cells was significantly higher compared to controls, suggesting that adiponectin rendered the olfactory neurons more responsive to an odorant stimulus. To analyze whether the augmented responsiveness of sensory neurons was strong enough to elicit a higher neuronal activity in the olfactory bulb, the number of activated periglomerular cells of a distinct glomerulus was determined by monitoring the stimulus-induced expression of c-fos. The studies were performed using the transgenic mOR256-17-IRES-tauGFP mice which allowed to visualize the corresponding glomerulus and to stimulate with a known ligand. The data indicate that upon exposure to 2,3-hexanedione in adiponectin-treated mice the number of activated periglomerular neurons was significantly increased compared to controls. The results of this study indicate that adiponectin increases the responsiveness of the olfactory system, probably due to a higher responsiveness of olfactory sensory neurons.     

Gal-NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system

A new monoclonal antibody, 2E11, was produced by immunizing mice with the microsomal fraction of rat accessory olfactory bulb cells. This IgM recognizes a previously described complex alpha-galactosyl containing glycolipid, as well as N-linked glycoproteins at 170 and 210 kD. These proteins correspond to a new nerve cell adhesion molecule (NCAM) glycoform, Gal-NCAM, which contains a blood group B-like oligosaccharide. During embryonic development, the 2E11 epitope is expressed by a subset of mature olfactory sensory neurons randomly dispersed throughout the olfactory epithelium, whereas in the olfactory bulb, immunostaining is restricted to medial areas of the nerve layer. When compared to PSA-NCAM, another NCAM glycoform, Gal-NCAM has a mutually exclusive distribution pattern both in the olfactory epithelium and in the olfactory bulb. We propose a model for the hierarchy of neuronal maturation in the olfactory epithelium, including a switch from PSA-NCAM expression by immature neurons to the expression of Gal-NCAM by mature neurons.     

Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis.     

Gal‐NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system

A new monoclonal antibody, 2E11, was produced by immunizing mice with the microsomal fraction of rat accessory olfactory bulb cells. This IgM recognizes a previously described complex α‐galactosyl containing glycolipid, as well as N‐linked glycoproteins at 170 and 210 kD. These proteins correspond to a new nerve cell adhesion molecule (NCAM) glycoform, Gal‐NCAM, which contains a blood group B‐like oligosaccharide. During embryonic development, the 2E11 epitope is expressed by a subset of mature olfactory sensory neurons randomly dispersed throughout the olfactory epithelium, whereas in the olfactory bulb, immunostaining is restricted to medial areas of the nerve layer. When compared to PSA‐NCAM, another NCAM glycoform, Gal‐NCAM has a mutually exclusive distribution pattern both in the olfactory epithelium and in the olfactory bulb. We propose a model for the hierarchy of neuronal maturation in the olfactory epithelium, including a switch from PSA‐NCAM expression by immature neurons to the expression of Gal‐NCAM by mature neurons. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 173–185, 2000     

Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit

Axons of olfactory sensory neurons expressing a given odorant receptor converge to a few glomeruli in the olfactory bulb. We have generated mice with unresponsive olfactory sensory neurons by targeted mutagenesis of a cyclic nucleotide-gated channel subunit gene, OCNC1. When these anosmic mice were crossed with mice in which neurons expressing a given odorant receptor can be visualized by coexpression of an axonal marker, the pattern of convergence was affected for one but not another receptor. In a novel paradigm, termed monoallelic deprivation, axons from channel positive or negative neurons that express the same odorant receptor segregate into distinct glomeruli within the same bulb. Thus, the peripheral olfactory projections are in part influenced by mechanisms that depend on neuronal activity.     

Olfactory epithelia differentially express neuronal markers

All three olfactory epithelia, the olfactory epithelium proper (OE), the septal organ of Masera (SO), and the vomeronasal organ of Jacobson (VNO) originate from the olfactory placode. Here, their diverse neurochemical phenotypes were analyzed using the immunohistochemical expression pattern of different neuronal markers. The olfactory bulb (OB) served as neuronal control. Neuronal Nuclei Marker (NeuN) is neither expressed in sensory neurons in any of the three olfactory epithelia, nor in relay neurons (mitral/tufted cells) of the OB. However, OB interneurons (periglomerular/granule cells) labeled, as did supranuclear structures of VNO supporting cells and VNO glands. Protein Gene Product 9.5 (PGP9.5 = C-terminal ubiquitin hydrolase L1 = UCHL1) expression is exactly the opposite: all olfactory sensory neurons express PGP9.5 as do OB mitral/tufted cells but not interneurons. Neuron Specific Enolase (NSE) expression is highest in the most apically located OE and SO sensory neurons and patchy in VNO. In contrast, the cytoplasm of the most basally located neurons of OE and SO immunoreacted for Growth Associated Protein 43 (GAP-43/B50). In VNO neurons GAP-43 labeling is also nuclear. In the cytoplasm, Olfactory Marker Protein (OMP) is most intensely expressed in SO, followed by OE and least in VNO neurons; further, OMP is also expressed in the nucleus of basally located VNO neurons. OB mitral/tufted cells express OMP at low levels. Neurons closer to respiratory epithelium often expressed a higher level of neuronal markers, suggesting a role of those markers for neuronal protection against take-over. Within the VNO the neurons show clear apical–basal expression diversity, as they do for factors of the signal transduction cascade. Overall, expression patterns of the investigated neuronal markers suggest that OE and SO are more similar to each other than to VNO.     

Neuronal degeneration and regeneration induced by axotomy in the olfactory epithelium of Xenopus laevis

The olfactory epithelium (OE) has the remarkable capability to constantly replace olfactory receptor neurons (ORNs) due to the presence of neural stem cells (NSCs). For this reason, the OE provides an excellent model to study neurogenesis and neuronal differentiation. In the present work, we induced neuronal degeneration in the OE of Xenopus laevis larvae by bilateral axotomy of the olfactory nerves. We found that axotomy induces specific‐ neuronal death through apoptosis between 24 and 48h post‐injury. In concordance, there was a progressive decrease of the mature‐ORN marker OMP until it was completely absent 72h post‐injury. On the other hand, neurogenesis was evident 48h post‐injury by an increase in the number of proliferating basal cells as well as NCAM‐180– GAP‐43+ immature neurons. Mature ORNs were replenished 21 days post‐injury and the olfactory function was partially recovered, indicating that new ORNs were integrated into the olfactory bulb glomeruli. Throughout the regenerative process no changes in the expression pattern of the neurotrophin Brain Derivate Neurotrophic Factor were observed. Taken together, this work provides a sequential analysis of the neurodegenerative and subsequent regenerative processes that take place in the OE following axotomy. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1308–1320, 2017     

Loss of STOP Protein Impairs Peripheral Olfactory Neurogenesis

Background

STOP (Stable Tubulin-Only Polypeptide) null mice show behavioral deficits, impaired synaptic plasticity, decrease in synaptic vesicular pools and disturbances in dopaminergic transmission, and are considered a neurodevelopmental model of schizophrenia. Olfactory neurons highly express STOP protein and are continually generated throughout life. Experimentally-induced loss of olfactory neurons leads to epithelial regeneration within two months, providing a useful model to evaluate the role played by STOP protein in adult olfactory neurogenesis.

Methodology/Principal Findings

Immunocytochemistry and electron microscopy were used to study the structure of the glomerulus in the main olfactory bulb and neurogenesis in the neurosensorial epithelia. In STOP null mice, olfactory neurons showed presynaptic swellings with tubulovesicular profiles and autophagic-like structures. In olfactory and vomeronasal epithelia, there was an increase in neurons turnover, as shown by the increase in number of proliferating, apoptotic and immature cells with no changes in the number of mature neurons. Similar alterations in peripheral olfactory neurogenesis have been previously described in schizophrenia patients. In STOP null mice, regeneration of the olfactory epithelium did not modify these anomalies; moreover, regeneration resulted in abnormal organisation of olfactory terminals within the olfactory glomeruli in STOP null mice.

Conclusions/Significance

In conclusion, STOP protein seems to be involved in the establishment of synapses in the olfactory glomerulus. Our results indicate that the olfactory system of STOP null mice is a well-suited experimental model (1) for the study of the mechanism of action of STOP protein in synaptic function/plasticity and (2) for pathophysiological studies of the mechanisms of altered neuronal connections in schizophrenia.     

Receptor guanylyl cyclases in mammalian olfactory function

The contributions of guanylyl cyclases to sensory signaling in the olfactory system have been unclear. Recently, studies of a specialized subpopulation of olfactory sensory neurons (OSNs) located in the main olfactory epithelium have provided important insights into the neuronal function of one receptor guanylyl cyclase, GC-D. Mice expressing reporters such as β-galactosidase and green fluorescent protein in OSNs that normally express GC-D have allowed investigators to identify these neurons in situ, facilitating anatomical and physiological studies of this sparse neuronal population. The specific perturbation of GC-D function in vivo has helped to resolve the role of this guanylyl cyclase in the transduction of olfactory stimuli. Similar approaches could be useful for the study of the orphan receptor GC-G, which is expressed in another distinct subpopulation of sensory neurons located in the Grueneberg ganglion. In this review, we discuss key findings that have reinvigorated the study of guanylyl cyclase function in the olfactory system.     

Morphological and electrophysiological examination of olfactory sensory neurons during the early developmental prolarval stage of the sea lamprey Petromyzon marinus L

This study examined olfactory sensory neuron morphology and physiological responsiveness in newly hatched sea lamprey, Petromyzon marinus L. These prolarvae hatch shortly after neural tube formation, and stay within nests for approximately 18 days, before moving downstream to silty areas where they burrow, feed and pass to the larval stage. To explore the possibility that the olfactory system is functioning during this prolarval stage, morphological and physiological development of olfactory sensory neurons was examined. The nasal cavity contained an olfactory epithelium with ciliated olfactory sensory neurons. Axons formed aggregates in the basal portion of the olfactory epithelium and spanned the narrow distance between the olfactory epithelium and the brain. The presence of asymmetric synapses with agranular vesicles within fibers in the brain, adjacent to the olfactory epithelium suggests that there was synaptic connectivity between olfactory sensory axons and the brain. Neural recordings from the surface of the olfactory epithelium showed responses following the application of L-arginine, taurocholic acid, petromyzonol sulfate (a lamprey migratory pheromone), and water conditioned by conspecifics. These results suggest that lampreys may respond to olfactory sensory input during the prolarval stage.     

CNS glial cells support in vitro survival, division, and differentiation of dissociated olfactory neuronal progenitor cells.

Olfactory receptor neurons (ORNs) are replaced and differentiate in adult animals, but differentiation in dissociated cell culture has not been demonstrated. To test whether contact with the CNS regulates maturation, neonatal rat olfactory cells were grown on a culture substrate or on CNS astrocytes. Mature ORNs, immunopositive for olfactory marker protein (OMP), disappeared rapidly from both systems. Neurons positive for neuron-specific tubulin (immature and mature) disappeared from substrate-only cultures, but remained abundant in the cocultures. OMP-positive neurons reappeared after 10 days in vitro. Pulse labeling with [3H]thymidine showed extensive neurogenesis of both immature and mature olfactory neurons. This demonstrates, in vitro, both division and differentiation of olfactory progenitor cells.     

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