Search

To determine the morphological basis for the neurotrophic effects of brain-derived neurotrophic factor (BDNF) in the primary olfactory pathway (POP), tyrosine kinase receptor B (TrkB), a membrane-bound receptor for BDNF, was identified and localized in axons of olfactory receptor cells (ORC) of neonatal rat olfactory mucosa using immuno-histochemical and -cytochemical techniques. Initially, the immunospecificity of an anti-TrkB antibody that had been used as a specific antibody for full-length TrkB was confirmed in the olfactory mucosa. Then, a combination of a reduced osmium-LR-White and post-embedding immunogold technique was applied to ORC axons in the lamina propria just beneath the olfactory epithelium. Immunogold particles, which indicate TrkB immunoreactivity, were noted either in close association with the plasma membranes of ORC axons, and designated plasma-lemmal (PL), or within their cytoplasm, and designated cytoplasmic (CP). Most PL particles were seen in the CP portion of the axonal plasma membranes, suggesting that the anti-TrkB antibody binds to the membrane-inserted TrkB that acts as a functional receptor. Some CP particles were on vesicular structures. Quantitative analysis demonstrated that the ratio of CP to PL particles was 7:3, and this ratio was constant between animals examined (n = 5). Because membrane proteins are wrapped in vesicles and transported within the axonal cytoplasm and inserted into the plasma membrane to function there, the present study suggests that TrkB is transported within the cytoplasm of ORC axons and is positioned as a functional receptor for BDNF in their membranes.

The vomeronasal organ comprises a pair of narrow tubes in the mammalian nasal septum, serving as a chemosensory system for pheromones. We examined the expression and localization of water channel aquaporins (AQPs) in the rat vomeronasal organ. AQP1 was localized in blood vessels, being particularly abundant in cavernous tissues of the nonsensory mucosa. AQP5 was found in the apical membrane of the gland acinar cells in the vomeronasal organ. AQP3 was detected in the basal cells of the nonsensory epithelium, whereas it was absent in the sensory epithelium. AQP4 was found in both the sensory and the nonsensory epithelia. Interestingly, AQP4 was highly concentrated in the sensory cells of the sensory epithelium. Immunoelectron microscopic examination clearly showed that AQP4 was localized at the plasma membrane in the cell body and lateral membrane of the dendrite, except for the microvillous apical membrane. Nerve fiber bundles emanating from neuronal sensory cells were positive for AQP4, whereby the plasma membrane of each axon was positive for AQP4. These observations clearly show that neuronal sensory cells in the vomeronasal organ are unique in that they express abundant AQP4 at their plasma membrane. This is in marked contrast to the olfactory and central nervous systems, where AQPs are not detectable in neurons, and instead, AQP4 is abundant in the supporting cells and astrocytes surrounding them. The present findings suggest a unique water-handling feature in neuronal sensory cells in the vomeronasal organ.

Although several lines of evidence have suggested that sex steroids influence olfaction, little is known about the cellular basis of steroid-metabolizing enzymes in the olfactory system. Thus, we aimed to examine gene expression and immunolocalization of four sex steroid-metabolizing enzymes in the olfactory mucosa (OM) of albino rats; steroid side chain-cleaving enzyme (P450scc), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD-1), 17β-HSD type 2 (17β-HSD-2), and aromatase. P450scc is known to catalyze conversion from cholesterol to pregnenolone. 17β-HSD-1 catalyzes conversion from estrone to estradiol, and 17β-HSD-2 does the reverse. Aromatase catalyzes the conversion from testosterone to estradiol-17β. Messenger (m) RNAs of all four enzymes mentioned above were detected in the OM. Western blot analysis demonstrated that P450scc, 17β-HSD-1, and 17β-HSD-2 were detected in the OM. Immunoreactivity for these three enzymes was observed in sustentacular cells of the olfactory epithelium and acinar cells of Bowman's glands. Immunoelectron microscopy analysis demonstrated immunoreactivity for P450scc in mitochondria, and for 17β-HSD-1 and 17β-HSD-2 in the well-developed smooth endoplasmic reticulum and myeloid bodies of the sustentacular cells. The present study suggests that sustentacular cells and acinar cells of the Bowman's glands in the rat OM express at least three of the steroid-metabolizing enzymes, that is, P450scc 17β-HSD-1, and 17β-HSD-2, and de novo synthesis of estradiol takes place in the OM. Anat Rec, 300:402-414, 2017. © 2016 Wiley Periodicals, Inc.

The nervous cells in the brain and the peripheral nerves are isolated from the external environment by the blood-brain, blood-cerebrospinal fluid and blood-nerve barriers. The glucose transporter GLUT1 mediates the specific transfer of glucose across these barriers. The olfactory system is unique in that its sensory cells, olfactory receptor neurons, are embedded in the nasal olfactory epithelium and send their axons directly to the olfactory bulb of the brain. Only the apical parts of the olfactory receptor neurons are exposed to the lumen, and these serve as sensors for smell. Immunohistochemical examination showed that the tight junction protein occludin was present in the junctions of the olfactory epithelium. Endothelial cells in the blood vessels in the lamina propria of the olfactory mucosa were also positive for occludin. These observations suggest that the olfactory system is guarded from both the external environment and the blood. GLUT1 was abundant in these occludin-positive endothelial cells, suggesting that GLUT1 may serve in nourishing the cells of the olfactory system. Taken together, GLUT1 and occludin may serve as part of the machinery for the specific transfer of glucose in the olfactory system while preventing the non-specific entry of substances.

Two G protein subfamilies, Go(alpha) and Gi(alpha 2), were identified and localized immunohistochemically in the vomeronasal organ (VNO) of 5-month-old human fetuses. Immunoreactivity for Go(alpha) and Gi(alpha 2) was present in a subset of vomeronasal epithelial cells. Prominent immunoreactivity was observed in apical processes and their apical terminals facing onto the vomeronasal lumen. Nerve fibers associated with the VNO exhibited intense immunoreactivity for Go(alpha) and weak immunoreactivity for Gi(alpha 2). Since Go(alpha) and Gi(alpha 2) are characteristically expressed and coupled with putative pheromone receptors in rodent vomeronasal receptor neurons, the present results suggest the possibility that vomeronasal epithelial cells containing Go(alpha) and Gi(alpha 2) in human fetuses are chemosensory neurons.

The density of protein gene product 9.5- and tyrosine hydroxylase-immunoreactive nerve fibers innervating circumvallate papillae of the tongue was substantially increased in transgenic mice that overexpressed nerve growth factor (NGF) when compared with age-matched controls. The fiber density was age-dependent. Only transgenic mice contained NGF-immunoreactive basal cells in the vicinity of taste buds, indicating that target-derived NGF induced novel hyperinnervation of the circumvallate papillae.

The localization of alpha-D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine sugar residues of glycoconjugates in the vomeronasal organ, olfactory mucosa, and septal organ in the nasal mucosae of rats was investigated using lectinohistochemical techniques combined with bright-field, epifluorescence, and confocal laser scanning microscopy. Glycoconjugates in the mucomicrovillar complex of the vomeronasal organ contained all the sugar residues investigated, whereas glycoconjugates in the mucociliary complex of the olfactory mucosa and septal organ contained only N-acetyl-D-glucosamine. Vomeronasal receptor neurons expressed glycoconjugates with terminal alpha-D-galactose and beta-N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine residues, whereas olfactory and septal receptor neurons expressed glycoconjugates with only N-acetyl-D-glucosamine residues. Secretory granules of glands of the vomeronasal organ contained glycoconjugates with terminal alpha-D-galactose and N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine, whereas those of the Bowman's glands and glands of septal organ contained glycoconjugates with only internal N-acetyl-D-glucosamine residues. The results demonstrate that the glycoconjugates expressed by vomeronasal receptor neurons and glands contain terminal alpha-D-galactose and beta-N-acetyl-D-galactosamine sugar residues that are not expressed by analogous cells in the olfactory mucosa and septal organ.

We examined the binding sites of Bandeiraea simplicifolia lectin I (BSL-I) and Vicia villosa agglutinin (VVA) which bind to the vomeronasal nerve of the rat. BSL-I stained the whole vomeronasal nerve and glomerular layers. VVA strongly stained the posterior 2/3, but weakly stained the anterior 1/3 of the glomerular layer. These results indicate that the glomeruli of the rat AOB have two subdivisions revealed by lectin histochemistry using BSL-I and VVA.

GABA-immunoreactive (IR) neurons were observed in the accessory olfactory bulb (AOB) of adult female rats. Many somata and dendritic trees of periglomerular-located cells were GABA-IR and the size of their somata was variable. Numerous somata and dendrates in the granule cell layer were IR. These results suggest that a large number of the interneurons in the AOB are GABA-IR.

Mitral/tufted cells (MTCs) of the accessory olfactory bulb (AOB) of adult rats were investigated light microscopically with the rapid Golgi method. The somata of the MTCs, appearing ovoid or triangular in shape, are distributed throughout the external plexiform layer. The soma size varies from small to large (12-26 microns). Apical dendrites originating from the soma enter the glomerular layer to provide branches that form the glomerular arbors. After making a glomerular arbor, some dendrites develop a second arbor (en passant and terminal arbors, respectively). The MTCs have a very diverse dendritic branching pattern and most have a variable number of glomerular arbors per cell (up to 6); we have tentatively classified the MTCs into simple, intermediate, and complex. Of the glomerular arbors, 80% have a diameter of less than 50 microns. The glomerular arbors have been classified as baskets (small spherical or ovoid) with short loopy processes; balls of yarn (large and nearly spherical) with loosely intermingled thick loops; and bushes (small to large and rather polymorphic) with irregular processes. The MTCs send dendritic arbors to terminate in one or more glomeruli where they are arranged in several different types of endings. Since it is generally believed that the dendrites of mitral and tufted cells of the main olfactory bulb terminate in only one glomerulus, the difference in the termination of the dendrites of the MTCs may represent a morphological characteristic that is relevant to the coding and/or integration of sensory information.

Using an immunoperoxidase technique, somatostatin-28-like immunoreactive (LIR) neurons were observed in the main and accessory olfactory bulb (MOB and AOB) of adult rats. In the MOB, a restricted population of periglomerular cells in the glomerular layer, some superficial short-axon cells in the juxtaglomerular layer, and some deep short-axon cells in the granule cell layer were IR. The periglomerular and the superficial short-axon cells were stained so well that they looked like Golgi-impregnated specimens. In the AOB, a very small population of small neurons in the glomerular layer, a very few medium-sized and large neurons in the external plexiform layer, and some neurons in the granule cell layer, which seem to be corresponding to the deep short-axon cells in the MOB, were IR. The present results have revealed that different morphological types of bulbar neurons are somatostatin-28-LIR; they also indicate neurochemical differences between the MOB and AOB.

Using the rapid Golgi method, the accessory olfactory bulb (AOB) of the adult rat was examined. The principal neurons of the AOB were very variable in terms of their dendritic branching pattern and morphology of the glomerular arbors. Some glomerular arbors occupied small portions, other large portions of a single glomerulus. These morphological patterns suggest that complex integrative events occur in the AOB.

Binding sites of Griffonia simplicifolia I-B4 isolectin (GS-I-B4), which recognizes terminal alpha-galactose residues of glycoconjugates, were examined in the juxtaluminal region of the rat vomeronasal sensory epithelium and its associated glands of the vomeronasal organ, using a lectin cytochemical technique. Lowicryl K4M-embedded ultra-thin sections, which were treated successively with biotinylated GS-I-B4 and streptavidin-conjugated 10 nm colloidal gold particles, were observed under a transmission electron microscope. Colloidal gold particles, which reflect the presence of terminal alpha-galactose-containing glycoconjugates, were present in vomeronasal receptor neurons in the sensory epithelium and secretory granules of acinar cells of associated glands of the epithelium. Quantitative analysis demonstrated that the density of colloidal gold particles associated with sensory cell microvilli that projected from dendritic endings of vomeronasal neurons was considerably higher than that of microvilli that projected from neighboring sustentacular cells. The same was true for the apical cytoplasms of these cells just below the microvilli. These results suggest that of the sensory microvilli and dendritic endings contained a much larger amount of the alpha-galactose-containing glycoconjugates, compared with those in sustentacular microvilli. Further, biochemical analyses demonstrated several vomeronasal organ-specific glycoproteins with terminal alpha-galactose.

Brain-derived neurotrophic factor (BDNF) is one of the neurotrophins, and known to facilitate differentiation, growth and maturation of neurons. BDNF binds to the high-affinity receptor, tyrosine kinase receptor B (TrkB) to initiate signal transduction (Korsching, 1993; Lindsay et al., 1994). Olfactory and vomeronasal receptor neurons (ORNs and VRNs, respectively) are chemoreceptors located in the nasal cavity of most mammalian species (Graziadei, 1977). In the olfactory epithelium (OE) lining the olfactory mucosa (OM), progenitor cells differentiate into immature and mature ORNs; a single dendrite extending from the apical pole of each soma reaches the surface of the OE to form a dendritic ending, and a single axon from the base of the soma runs downward to pass through the basement membrane of the OE to reach the brain. Similar bipolar neurons are also contained in the vomeronasal sensory epithelium (VSE) of the vomeronasal organ (VNO), and designated as VRNs or vomeronasal sensory cells/ neurons (Graziadei, 1977; Takami, 2002). Although several neurotrophic factors including neurotrophins are thought to be involved in the development, maturation, and regeneration of ORNs (Carter and Roskams, 2002; Schwob, 2002), the functional roles of BDNF remain to be understood. In the case of the VRNs, we were the first to report the distribution of BDNF and TrkB at both domestic (Takami et al., 2001) and international (Takami and Nishiyama, 1997b) meetings. In this paper, we present summary of our recent studies concerning BDNF and TrkB in the rat OM and VNO. Our overall research goal is to clarify functional roles of BDNF in the differentiation and maturation of ORNs and VRNs.

The organization of the mucomicrovillar complex of the vomeronasal sensory epithelium of adult rats was examined using confocal laser scanning microscopy. In specimens labeled with the FITC-conjugated isolectin B4 of Bandeiraea simplicifolia, which recognizes terminal alpha-galactose sugar residues of glycoconjugates, we demonstrated that the mucomicrovillar complex was composed of islet-like structures with a high-density alpha-galactose core. The mucomicrovillar complex was further resolved into sensory and mucoid components in double-labeling and dual scanning experiments. The sensory component, which consists of the dendritic terminals of olfactory marker protein-immunoreactive vomeronasal receptor neurons, contained cytosolic glycoconjugates with terminal alpha-galactose sugar residues. The extracellular mucoid component consisted of glycoconjugates containing terminal alpha-galactose derived from the glands associated with the vomeronasal organ. These results demonstrated the complex microchemical organization of the sensory and mucoid components of the mucomicrovillar complex.