A functional map in rat olfactory epithelium

Multiple (four or eight) electrode arrays were placed for simultaneous electro-olfactogram (EOG) recordings of responses to a series of odors applied directly to the olfactory epithelium. Three different surfaces of the epithelium were exposed in rats immediately after death by anesthetic overdose. We tested three terpene compounds (carvone, limonene and 1,8-cineole) across the epithelium along the medial surface of the endoturbinate bones. Carvone, a ketone, evoked larger responses dorsally on the epithelium. The largest responses to 1,8-cineole (an ether) were seen in an intermediate-ventral region. The responses to limonene (a hydrocarbon) did not vary greatly across the regions, although they were often larger ventrally. The response distributions deviated from this simple pattern on the caudal part of endoturbinate IV, where the carvone responses were small and the limonene responses were larger. These differences were evident across a substantial concentration range. Similar distributions were seen for these three odors in tests along the dorsal-to-ventral direction across the nasal septum and in the medial-to-lateral direction across the dorsal aspect of one of the endoturbinate bones reaching out into the lateral recess. We argue that the spatial distributions of responses are correlated with the olfactory receptor gene expression zones.     

Internalization of odorant-binding proteins into the mouse olfactory epithelium

The detection of odorants in vertebrates is mediated by chemosensory neurons that reside in the olfactory epithelium of the nose. In land-living species, the hydrophobic odorous compounds inhaled by the airstream are dissolved in the nasal mucus by means of specialized globular proteins, the odorant-binding proteins (OBPs). To assure the responsiveness to odors of each inhalation, a rapid removal of odorants from the microenvironment of the receptor is essential. In order to follow the fate of OBP/odorant complexes, a recombinant OBP was fluorescently labeled, loaded with odorous compounds, and applied to the nose of a mouse. Very quickly, labeled OBP appeared inside the sustentacular cells of the epithelium. This uptake occurred only when the OBP was loaded with appropriate odorant compounds. A search for candidate transporters that could mediate such an uptake process led to the identification of the low density lipoprotein receptor Lrp2/Megalin. In the olfactory epithelium, megalin was found to be specifically expressed in sustentacular cells and the Megalin protein was located in their microvilli. In vitro studies using a cell line that expresses megalin revealed a rapid internalization of OBP/odorant complexes into lysosomes. The uptake was blocked by a Megalin inhibitor, as was the internalization of OBPs into the sustentacular cells of the olfactory epithelium. The results suggest that a Megalin-mediated internalization of OBP/odorant complexes into the sustentacular cells may represent an important mechanism for a rapid and local clearance of odorants.     

Microwave effect on camphor binding to rat olfactory epithelium

Microwave radiation decreased specific camphor binding to a membrane fraction of rat epithelium but not to a Triton X-100 extract of this fraction. Inhibition of the ligand binding did not depend on the modulation frequency of the microwave field in the region 1-100 Hz and was not a linear function of specific absorption rate (SAR). The decreased ligand binding was due to a shedding or release of the specific camphor-binding protein from the membrane into solution. It is highly probable that several other membrane proteins may be shed into solution during microwave exposure.     

The ultrastructure of the white rat olfactory epithelium]

As a result of electron microscope observation of olfactory mucosa in the white rat, variability of structure of the supporting cells was established. Among these cells secretory ones and those rich in endoplasmic reticulum were discovered. The differences revealed in dendrites and rods of olfactory cells seem to be associated with functional condition of these cells.     

Differential localization of G proteins, Gi and Go, in the olfactory epithelium and the main olfactory bulb of the rat.

The immunohistochemical localization of proteins Gi1 (plus Gi3). Gi2 and Go was studied in the olfactory epithelium and the main olfactory bulb of rats, using purified antibodies to the respective alpha subunits and beta gamma subunits of these G proteins. In the olfactory epithelium, only a restricted population of olfactory cells was immunopositive for Gi2 alpha, but others were not. The immunoreactivity for Gi1 alpha/Gi3 alpha was not observed. The olfactory epithelium was immunopositive for both Go alpha and beta gamma, but its apical surface was immunopositive only for beta gamma. In the main olfactory bulb, all layers were intensely immunopositive for Go alpha and beta gamma but weakly for Gi2 alpha. In contrast to the negative or weak immunostainings in the olfactory nerve fiber layer and glomeruli, the molecular and the internal granular layers were intensely immunopositive for Gi1 alpha/Gi3 alpha. These findings suggest the functional difference among Gi1/Gi3, Gi2 and Go in the signal transduction in the olfactory system.     

The subunits of specific odor-binding glycoproteins from rat olfactory epithelium

The specific odor-binding glycoproteins have been isolated from rat olfactory epithelium. They consist of two subunits, gp88 and gp55. Subunit gp88 is capable of odorant binding.     

Identification in the rat olfactory epithelium new subgroup YM-1 chitinase-like protein

Novel protein with a molecular mass of ~43 kDa from rat olfactory epithelium in pathophysiological conditions was discovered. Its amino acid sequence and affiliation with the family 18 glycohydrolase subgroup of chitinase-like proteins YM-1 were determined.     

Calcium-binding proteins: differential expression in the rat olfactory cortex after neonatal olfactory bulbectomy

Calbindin, parvalbumin, and calretinin, members of EF-hand calcium-binding proteins, play important roles in buffering intracellular calcium ions. These proteins are localized in distinct populations of cells in the olfactory bulb (the primary sensory relay in the olfactory system) and its major synaptic target, the primary olfactory cortex (POC). In the present study, the postnatal expression of these calcium-binding proteins in layer III of POC was quantitatively examined 30 days after neonatal bulbectomy, a manipulation known to cause cell death and neurotransmitter changes. The numbers of both calbindin and parvalbumin-immunoreactive profiles showed significant increases (68% and 163%, respectively), while calretinin-immunoreactive profiles exhibited a 46% reduction. The data demonstrate that the expression of these calcium-binding proteins is regulated in part by the afferent input from the olfactory bulb. Furthermore, the resultant increase in calbindin and parvalbumin expression may provide neuroprotective support necessitated by possible alterations in intracellular calcium ions and other neurochemical factors that accompany neonatal bulb removal.     

Expression of the cellular retinoic acid binding proteins,type II and type I,in mature rat olfactory epithelium

Cellular retinoic acid binding proteins, types I and II (CRABP I and II), are cytosolic proteins that exhibit a binding preference for all-trans retinoic acid. As part of a larger study to determine whether retinoic acid plays a role in neurogenesis in vivo, we questioned whether CRABP II is present in rat postnatal olfactory epithelium (OE), a sensory tissue that continually replaces neurons throughout adult life. We have determined that both CRABP II and CRABP I proteins and the mRNAs that encode them are present in postnatal rat OE. Immunoreactivity with CRABP II and CRABP I antibodies was not observed in the nasal respiratory epithelium. Double immunolabeling experiments, conducted with antibodies showing specificity for each antigen, indicate that CRABP II and CRABP I are found in different cell types within the olfactory neuroepithelium. We also asked whether CRABP II is expressed in the postnatal rat retina, a neural tissue that is not known to show neuron replacement during adult life. CRABP type II immunoreactivity was not observed in the mature rat retina. The presence of CRABP II in postnatal OE and its absence from mature retina is consistent with previous reports indicating that the distribution of CRABP II in adult mammals is restricted to tissue systems that exhibit ongoing growth and differentiation throughout life.     

3H-estradiol binding to cytosol receptors of the rat olfactory epithelium

Two types of cytosol receptors of 3H-estradiol with high affinity and limited quantity of binding sites (KDI = 1-2 nM, BmaxI = 8 fmoles/mg protein; KDII = 10 nM, BmaxII = 8 fmoles/mg protein) were determined in the rat olfactory tissue. The amount of high affinity receptors of type I does not change with maturation of the rats, and has no sex difference. The role of estradiol receptors in the olfactory tissue of the rats is discussed.     

Renal cystic disease proteins play critical roles in the organization of the olfactory epithelium

It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases - polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) - localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (G(olf), AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia.     

Biopsies of human olfactory epithelium

It has been shown that olfactory epithelium can be safely biopsied from the living, intact human being. Observations of the ultrastructure of this epithelium shows changes that can then be correlated with the etiology and degree of olfactory loss, allowing a greater understanding of both normal transduction and of the pathology of dysfunction. Examples of the common forms of olfactory dysfunction are presented and discussed. Additionally, the technique will allow additional immuno-histochemical and molecular study of the tissue, will increase the understanding of both normal and pathological function and should translate to new therapeutic regimens.     

TRPC3-interacting triadic proteins in skeletal muscle

The expression of TRPC3 (canonical-type transient receptor potential cation channel type 3) is tightly regulated during skeletal muscle cell differentiation, and a functional interaction between TRPC3 and RyR1 [(ryanodine receptor type 1), an SR (sarcoplasmic reticulum) Ca2+-release channel] regulates the gain of SR Ca2+ release during EC (excitation-contraction) coupling. However, it has not been possible to demonstrate direct protein-protein interactions between TRPC3 and RyR1. To identify possible candidate(s) for a linker protein(s) between TRPC3 and RyR1 in skeletal muscle, in the present study we performed MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of a cross-linked triadic protein complex from rabbit skeletal triad vesicles and co-immunoprecipitation assays using primary mouse skeletal myotubes. From these studies, we found that six triadic proteins, that are known to regulate RyR1 function and/or EC coupling [TRPC1, JP2 (junctophilin 2), homer, mitsugumin 29, calreticulin and calmodulin], interacted directly with TRPC3 in a Ca2+-independent manner. However we again found no direct interaction between TRPC3 and RyR1. TRPC1 was identified as a potential physical link between TRPC3 and RyR1, as it interacted with both TRPC3 and RyR1, and JPs showed subtype-specific interactions with both RyR1 and TRPC3 (JP1-RyR1 and JP2-TRPC3). These results support the hypothesis that TRPC3 and RyR1 are functionally engaged via linker proteins in skeletal muscle.     

An ultrastructural and immunohistological study of the rat olfactory epithelium: Unique properties of olfactory sensory cells

The olfactory epithelium contains three cell types: basal cells, supporting cells and sensory neurons. Electron microscopy as well as immunofluorescence microscopy with intermediate-filament antibodies were used to study the rat olfactory epithelium in order to obtain more information about these different cell types and to try to investigate their histogenetic origins. We found mitoses in the basal-cell layer, as well as multiple centrioles and tonofilaments in some basal cells. As revealed by electron microscopy, the supporting cells contained tonofilaments and reacted strongly with antibodies to keratin, in line with their known epithelial nature. When antibodies to other intermediate-filament types were used, i.e. glial fibrillary acidic protein, vimentin, desmin and neurofilaments, no reaction was seen in the cells of the olfactory epithelium, with the exception of occasional staining of a few axons in the subepithelial layer by neurofilament antibodies. In particular, the cell bodies, dendrites and most axons of the sensory neurons were negative for a variety of antibodies against neurofilaments. Olfactory sensory neurons therefore belong to the very few cells in adult animals which seem to lack intermediate filaments. We discuss whether this finding is related to the fact that these cells are also unique among neurons in that they are not permanent cells but constantly turn over.     

Olfaction and neurogenesis in the olfactory epithelium

Anosmia was experimentally produced in strain C57BL/6 laboratory mice by treatment with 1% zinc sulfate solution. Structural and functional changes taking place in the olfactory epithelium were investigated during this process and during reinstatement of olfaction. Isoamyl acetate, butyl acetate, and substances present in murine urine were used as olfactory stimuli. Response to these odorants was found to recover from zinc sulfate action at different rates. The highest (both relative and absolute) daily rise in amplitude response was that induced by isoamyl acetate and butyl acetate and lowest in the case of odors of biological origin. Response to olfactory stimuli recovered most rapidly in the areas of the epithelium where maximum response to the same stimuli had been seen in intact animals."Biopharmautomatica" Combined Research and Production Unit, Gor'kii. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 500–506, July–August, 1990.     

Molecular composition of tight and adherens junctions in the rat olfactory epithelium and fila

Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the fila olfactoria (FO). Immunofluorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1-5 and zonula occludens(ZO) proteins 1-3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, beta- and p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. Differences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with differential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha, beta- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest differential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role of AJ proteins in neuroplastic processes in the peripheral olfactory pathway.