Differential development of odorant receptor expression patterns in the olfactory epithelium: a quantitative analysis in the mouse septal organ

The rodent olfactory epithelium expresses more than 1000 odorant receptors (ORs) with distinct patterns, yet it is unclear how such patterns are established during development. In the current study, we investigated development of the expression patterns of different ORs in the septal organ, a small patch of olfactory epithelium predominantly expressing nine identified ORs. The presumptive septal organ first appears at about embryonic day 16 (E16) and it completely separates from the main olfactory epithelium (MOE) at about postnatal day 7 (P7). Using in situ hybridization, we quantified the densities of the septal organ neurons labeled by specific RNA probes of the nine abundant OR genes from E16 to postnatal 3 months. The results indicate that olfactory sensory neurons (OSNs) expressing different ORs have asynchronous temporal onsets. For instance, MOR256-17 and MOR236-1 cells are present in the septal organ at E16; however, MOR0-2 cells do not appear until P0. In addition, OSNs expressing different ORs show distinct developmental courses and reach their maximum densities at different stages ranging from E16 (e.g. MOR256-17) to 1 month (e.g. MOR256-3 and MOR235-1). Furthermore, early onset does not correlate with high abundance in adult. This study reveals a dynamic composition of the OSNs expressing different ORs in the developing olfactory epithelium.     

Activity-Dependent Modulation of Odorant Receptor Gene Expression in the Mouse Olfactory Epithelium

Activity plays critical roles in development and maintenance of the olfactory system, which undergoes considerable neurogenesis throughout life. In the mouse olfactory epithelium, each olfactory sensory neuron (OSN) stably expresses a single odorant receptor (OR) type out of a repertoire of ∼1200 and the OSNs with the same OR identity are distributed within one of the few broadly-defined zones. However, it remains elusive whether and how activity modulates such OR expression patterns. Here we addressed this question by investigating OR gene expression via in situ hybridization when sensory experience or neuronal excitability is manipulated. We first examined the expression patterns of fifteen OR genes in mice which underwent neonatal, unilateral naris closure. After four-week occlusion, the cell density in the closed (sensory-deprived) side was significantly lower (for four ORs), similar (for three ORs), or significantly higher (for eight ORs) as compared to that in the open (over-stimulated) side, suggesting that sensory inputs have differential effects on OSNs expressing different OR genes. We next examined the expression patterns of seven OR genes in transgenic mice in which mature OSNs had reduced neuronal excitability. Neuronal silencing led to a significant reduction in the cell density for most OR genes tested and thinner olfactory epithelium with an increased density of apoptotic cells. These results suggest that sensory experience plays important roles in shaping OR gene expression patterns and the neuronal activity is critical for survival of OSNs.     

Distribution of 1,25-Dihydroxyvitamin D3 Receptor Immunoreactivity in the Rat Olfactory System

1.The rat olfactory system contains numerous target sites for 1,25-dihydroxyvitamin D₃, as determined by receptor protein (VDR) immunocytochemistry and in situ hybidization.2.Nuclear and cytoplasmic VDR immunoreactivity as well as the corresponding hybridization signal was observed in neurons in the olfactory epithelium, the olfactory bulb, and throughout the limbic system in locations also known to be glucocorticoid targets.3.The widespread distribution of VDR indicates the distinct functional importance of 1,25-dihydroxyvitamin D₃ for olfactory perception.     

Characterizing the expression of the human olfactory receptor gene family using a novel DNA microarray

Background  

Olfactory receptor (OR) genes were discovered more than a decade ago, when Buck and Axel observed that, in rats, certain G-protein coupled receptors are expressed exclusively in the olfactory epithelium. Subsequently, protein sequence similarity was used to identify entire OR gene repertoires of a number of mammalian species, but only in mouse were these predictions followed up by expression studies in olfactory epithelium. To rectify this, we have developed a DNA microarray that contains probes for most predicted human OR loci and used that array to examine OR gene expression profiles in olfactory epithelium tissues from three individuals.     

Topographic patterns of odorant receptor expression in mammals: a comparative study

In a comparative study, molecular probes for various odorant receptor subtypes were employed in in situ hybridization experiments on tissue sections through the nose from different mammalian species. OR37 reactive neurons were found exclusively in the rodent species, where they were clustered in very similar position within the nasal cavities; an OR37-related receptor subtype was not detectable in the rabbit. All other subtypes tested, hybridized across species borders to neurons that were distributed within distinct zones of the olfactory epithelium. Most receptor types were found in the same zone in all species; however, a few subtypes which are expressed in the medial zone in rat were found in the dorsal zone in guinea pig.     

Adenovirus-mediated gene transfer in olfactory neurons in vivo

We used recombinant adenoviruses as a means of expressing exogenous genes in olfactory neurons in vivo. A replication incompetent adenovirus (type 5, Ad5) carrying the reporter gene lacZ, which codes for the enzyme β-galactosidase (β-Gal), was applied in solution to the olfactory epithelia of rats. The expression of lacZ was controlled by the cytomegalovirus immediate-early promoter/enhancer. β-Gal expression was observed 1 day postinfection and was maximal at 3–10 days, although it could be detected for at least 21 days postinfection. Expression patterns were heterogeneous, ranging from a few percent to over 25% of the cells in different regions of both turbinate and septal epithelium. Staining was stronger in the olfactory versus respiratory epithelia. In olfactory epithelium staining was almost entirely restricted to olfactory neurons. β-Gal staining was also observed in the olfactory axons so that nerve bundles could be traced to their targets in the glomerular layer of the olfactory bulb. Intense staining of some glomeruli was evident as long as 21 days postinfection. There was no evidence of cell loss or tissue damage due to viral infection. These results demonstrate that it is possible to use recombinant Ad5 for expressing foreign genes in olfactory neurons. This technique could be used in olfactory neurons to increase expression levels of olfactory specific genes, including the odor receptor, putative guidance and growth molecules, or elements of the transduction cascade, in order to elucidate their biological functions in vivo. © 1996 John Wiley & Sons, Inc.     

Androgen receptor mRNA expression in Xenopus laevis CNS: Sexual dimorphism and regulation in laryngeal motor nucleus

Using Northern analysis, in situ hybridization, and nuclease protection assays, the expression and regulation of androgen receptor messenger RNA (AR mRNA) was examined in the CNS of juvenile Xenopus laevis. Only one of the AR mRNA isoforms expressed in X. laevis is transcribed in the CNS as shown by Northern blot analysis. Nuclease protection assays demonstrate that the expression of AR mRNA is higher in the brain stem than in the telencephalon and diencephalon. Although expression of AR mRNA is widespread throughout the CNS, cells of cranial nerve nucleus IX-X (N. IX-X) and spinal cord display the highest in situ hybridization signals in their cytoplasm. Double labeling using horseradish peroxidase and digoxigenin labeled AR probes reveals that laryngeal and anterior spinal cord motor neurons express AR mRNA. More cells express AR mRNA in N. IX-X of males than of females. The number of AR expressing cells in N. IX-X decreases following gonadectomy in both sexes, and dihydrotestosterone (DHT) treatment for 1 month reverses this effect. Increased expression of AR mRNA in the brain of DHT treated animals is also apparent in nuclease protection assays. Sex differences in number of AR expressing cells and hormone regulation of AR mRNA expression in motor nuclei may influence neuromuscular systems devoted to sexually differentiated behaviors. © 1996 John Wiley & Sons, Inc.     

Olfactory metamorphosis in the coastal tailed frog Ascaphus truei (Amphibia,Anura, Leiopelmatidae)

The structure of the olfactory organ in larvae and adults of the basal anuran Ascaphus truei was examined using light micrography, electron micrography, and resin casts of the nasal cavity. The larval olfactory organ consists of nonsensory anterior and posterior nasal tubes connected to a large, main olfactory cavity containing olfactory epithelium; the vomeronasal organ is a ventrolateral diverticulum of this cavity. A small patch of olfactory epithelium (the “epithelial band”) also is present in the preoral buccal cavity, anterolateral to the choana. The main olfactory epithelium and epithelial band have both microvillar and ciliated receptor cells, and both microvillar and ciliated supporting cells. The epithelial band also contains secretory ciliated supporting cells. The vomeronasal epithelium contains only microvillar receptor cells. After metamorphosis, the adult olfactory organ is divided into the three typical anuran olfactory chambers: the principal, middle, and inferior cavities. The anterior part of the principal cavity contains a “larval type” epithelium that has both microvillar and ciliated receptor cells and both microvillar and ciliated supporting cells, whereas the posterior part is lined with an “adult‐type” epithelium that has only ciliated receptor cells and microvillar supporting cells. The middle cavity is nonsensory. The vomeronasal epithelium of the inferior cavity resembles that of larvae but is distinguished by a novel type of microvillar cell. The presence of two distinct types of olfactory epithelium in the principal cavity of adult A. truei is unique among previously described anuran olfactory organs. A comparative review suggests that the anterior olfactory epithelium is homologous with the “recessus olfactorius” of other anurans and with the accessory nasal cavity of pipids and functions to detect water‐borne odorants. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

Decline and recovery of olfactory receptor expression following unilateral bulbectomy

The effects of unilateral olfactory bulb ablation upon the odorant receptor expression were studied during the degeneration/regeneration process in the olfactory epithelium of adult rats. Using the in situ hybridization approach, we compared the time course of decay and recovery of expression for three different receptor subtypes (OR14, OR5, OR124). The number of neurons expressing receptor subtypes dramatically decreased in the olfactory epithelium on the lesioned side and reached a minimum at day 5 postsurgery. A progressive recovery was then observed from day 5 to day 15 postlesion, when a plateau was reached. Noticeable differences in the recovery level of receptor expression were observed according to the zonal patterning: the recovery level for neurons located in the lateral zone reached 70% of the control side value while the recovery levels in the dorsal and medial zones represented 35% and 53% of this value, respectively. Axotomy experiments suggest that zone-specific differences in receptor reexpression reported after bulbectomy might be related to the trophic influence of the olfactory bulb.     

Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high affinity interactions with odorant organic anions

We have characterized the expression of organic anion transporter 6, Oat6 (slc22a20), in olfactory mucosa, as well as its interaction with several odorant organic anions. In situ hybridization reveals diffuse Oat6 expression throughout olfactory epithelium, yet olfactory neurons laser-capture microdissected from either the main olfactory epithelium (MOE) or the vomeronasal organ (VNO) did not express Oat6 mRNA. These data suggest that Oat6 is expressed in non-neuronal cells of olfactory tissue, such as epithelial and/or other supporting cells. We next investigated interaction of Oat6 with several small organic anions that have previously been identified as odortype components in mouse urine. We find that each of these compounds, propionate, 2- and 3-methylbutyrate, benzoate, heptanoate, and 2-ethylhexanoate, inhibits Oat6-mediated uptake of a labeled tracer, estrone sulfate, consistent with their being Oat6 substrates. Previously, we noted defects in the renal elimination of odortype and odortype-like molecules in Oat1 knockout mice. The finding that such molecules interact with Oat6 raises the possibility that odorants secreted into the urine through one OAT-mediated mechanism (Eraly et al., JBC 2006) are transported through the olfactory mucosa through another OAT-mediated mechanism. Oat6 might play a direct or indirect role in olfaction, such as modulation of the availability of odorant organic anions at the mucosal surface for presentation to olfactory neurons or facilitation of delivery to a distal site of chemosensation, among other possibilities that we discuss.     

Retrograde labeling and fine structure of olfactory receptor neurons in cat sharks

Ciliated and microvillar olfactory receptor cells have been reported in many fish species, including teleosts and elasmobranchs. Morphological studies have suggested that microvillar cells are the only olfactory receptor cells in the elasmobranchs; however, there is no direct evidence for this hypothesis. Here we used a cat shark (Scyliorhinus torazame) to determine the cell type of the olfactory receptor cells in elasmobranchs. Retrograde labeling with a fluorescent dye, Dil, labeled only cells in the second layer from the surface of the olfactory epithelium, suggesting that ciliated cells located in the surface layer are not olfactory receptor cells. In addition, electron microscopic observation revealed that the labeled cells in the second layer have a thin dendritic knob extending from the cell body to the free surface of the epithelium. A part of the dendritic knob facing the mucous layer did not have ciliary structures. These results provide evidence that the aciliate cells are the only olfactory receptor cells in the cat shark olfactory organ.     

Goα expression in the vomeronasal organ and olfactory bulb of the tammar wallaby

The vomeronasal organ (VNO) detects pheromones via 2 large families of receptors: vomeronasal receptor 1, associated with the protein Giα2, and vomeronasal receptor 2, associated with Goα. We investigated the distribution of Goα in the developing and adult VNO and adult olfactory bulb of a marsupial, the tammar wallaby. Some cells expressed Goα as early as day 5 postpartum, but by day 30, Goα expressing cells were distributed throughout the receptor epithelium of the VNO. In the adult tammar, Goα appeared to be expressed in sensory neurons whose nuclei were mostly basally located in the vomeronasal receptor epithelium. Goα expressing vomeronasal receptor cells led to all areas of the accessory olfactory bulb (AOB). The lack of regionally restricted projection of the vomeronasal receptor cell type 2 in the tammar was similar to the uniform type, with the crucial difference that the uniform type only shows expression of Giα2 and no expression of Goα. The observed Goα staining pattern suggests that the tammar may have a third accessory olfactory type that could be intermediate to the segregated and uniform types already described.     

Cells expressing preproenkephalin mRNA in the rat pineal gland are not serotonin-producing pinealocytes: Evidence usingin Situ hybridization combined with immunocytochemistry for serotonin

Summary 1. Preproenkephalin (PPEnk) mRNA expressing cells have been identified in rat pineal gland using radioactivein situ hybridization histochemistry. 2. Approximately 7% of the cells in the pineal gland (7.5±0.86, mean ± 95% CI) express PPEnk mRNA. These cells are distributed throughout the pineal as either scattered single cells or small groups of cells with large round or oval nuclei. 3. Usingin situ hybridization combined with ABC immunocytochemistry for serotonin (5-HT) in the same pineal sections, the PPEnk mRNA labeling cells are found not to be serotonin-immunoreactive cells. These data indicate that the PPEnk mRNA is expressed in a certain discrete subpopulation of cells in the rat pineal gland and these cells are not serotonin-producing pinealocytes. 4. The physiologic role of PPEnk-derived peptides in the pineal remains unknown. It is possible that these peptides either are synthesized and secreted as hormones or act as pineal paracrine signals.     

Rostro-caudal patterning of receptor-expressing olfactory neurones in the rat nasal cavity

The rostro-caudal extent of odorant receptor expression zones in the rat olfactory epithelium was analysed by means of in situ hybridization. Three broad non-overlapping zones were identified that extended along almost the entire anterior-posterior axis; each zone was composed of several separate bands running anterior to posterior throughout the olfactory epithelium. Superimposed onto these broad zones was the expression area of a particular receptor subtype (OR37); it was restricted to a small region of the epithelial sheet with a high density of reactive neurones in the centre and declining numbers towards the periphery of the region. A quantitative evaluation of the reactive cells revealed that, despite their diferent distribution patterns, all receptor subtypes were expressed in an equal number of neurones.     

An improved bioluminescence‐based signaling assay for odor sensing with a yeast expressing a chimeric olfactory receptor

The goal of this work was to improve the bioluminescence‐based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast‐expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G‐protein α‐subunit (Gpa1p) with the olfactory‐specific Gα_olf, the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence‐based odor‐sensing system using OR‐expressing yeast. Biotechnol. Bioeng. 2012; 109: 3143–3151. © 2012 Wiley Periodicals, Inc.     

Functional Morphology of the Olfactory Organ in Spinachia spinachia (L.) (Teleostei,Gasterosteidae)

The functional morphology of the olfactory organ in Spinachia spinachia (L.), which has only a single nare, was studied by light microscopy, scanning electron microscopy, and experimental investigations. It was shown that only the incoming water passes over the olfactory epithelium. The device for ventilating this olfactory organ is an accessory ventilation sac activated by respiratory pressure changes in the buccal cavity. This one-way water current over the olfactory epithelium in a monotrematous olfactory organ was found to be possible because of the morphology of the olfactory organ combined with movements of the lateral wall of the olfactory organ and the nasal tube during respiration. The olfactory epithelium is divided into irregular islets. Both ciliated receptor cells and microvillous receptor cells are present.     

A multireceptor genetic approach uncovers an ordered integration of VNO sensory inputs in the accessory olfactory bulb

Pheromone detection by the vomeronasal organ (VNO) is thought to rely on activation of specific receptors from the V1R and V2R gene families, but the central representation of pheromone receptor activation remains poorly understood. We generated transgenic mouse lines in which projections from multiple populations of VNO neurons, each expressing a distinct V1R, are differentially labeled with fluorescent proteins. This approach revealed that inputs from neurons expressing closely related V1Rs intermingle within shared, spatially conserved domains of the accessory olfactory bulb (AOB). Mitral cell-glomerular connectivity was examined by injecting intracellular dyes into AOB mitral cells and monitoring dendritic contacts with genetically labeled glomeruli. We show that individual mitral cells extend dendrites to glomeruli associated with different, but likely closely related, V1Rs. This organization differs from the labeled line of OR signaling in the main olfactory system and suggests that integration of information may already occur at the level of the AOB.     

Histology and ultrastructure of the olfactory organ of the freshwater pulmonate Helisoma trivolvis

Summary The olfactory organ of Helisoma trivolvis is located on the surface of the body at the base of the cephalic tentacles. An evagination of skin, the olfactory plica, at the base of the tentacle extends over the olfactory organ dorsally. The epithelium of the olfactory organs contains unspecialized epithelial cells, ciliated epithelial cells, basal cells, mucous secretory cells, and sensory dendrites. The surface of the epithelium has a complex brush border of thick plasmatic processes, which branch to form several terminal microvillar twigs. Long slender cytoplasmic processes form a dense spongy layer among the plasmatic processes beneath the level of the terminal twigs. Bipolar primary sensory neurons clustered beneath the epithelium of the olfactory organ send dendrites through the epithelium to the free surface. Some sensory endings have a few short cilia, but most bear only microvilli. Cilia of sensory endings and epithelial cells extend beyond the brush border of the epithelium. Small axons arise from the perikarya of the sensory neurons and enter a branch of the olfactory nerve. HRP tracing indicates that the axons pass to the cerebral ganglion without interruption. Histochemical tests indicate that the sensory neurons are neither aminergic nor cholinergic.