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It has been known that androgens and estrogens, which are referred to as sex steroids, make many effects on two major nasal chemosensory mucosae such as olfactory mucosa and vomeronasal organ. Our studies conducted in rodents have demonstrated that two of the constituent cells in the olfactory mucosa, sustentacular cells and acinar cells in the associated glands of the olfactory mucosa, Bowman’s glands, express four different enzymes involved in the biosynthesis of estradiol-17β (E2). Furthermore, our ongoing study has shown that olfactory sensory cells contain immunoreactivity for an estrogen receptor (beta-type). In case of vomeronasal organ, vomeronasal sensory cells express two enzymes that catalyze conversion of E2 and estrone, and that of testosterone and androstenedione. In addition, vomeronasal sensory cells contain an estrogen receptor (alpha-type). These results strongly suggest that de novo synthesis of E2 and metabolism of E2 take place in the olfactory mucosa and vomeronasal organ, respectively. With special emphasis of subcellular characteristics of steroid-producing cells, such as presence of large amount of smooth endoplasmic reticulum and vesicular mitochondria, we will introduce our findings and present working hypotheses for E2 functions in the olfactory mucosa and 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 vomeronasal organ (VNO) of 5-month-old fetuses was examined immunohistochemically by the use of an antiserum to protein gene product 9.5 (PGP). The purpose was to identify if the human fetal VNO is lined by neuroepithelium. The PGP antiserum labeled abundant cells within the vomeronasal epithelium (VE), nerve fiber bundles in its lamina propria, and cells associated with these bundles. PGP-immunoreactive (ir) vomeronasal epithelial cells were classified into three subtypes. Type I cells, about 44% of the total cells observed, did not have any processes and tended to be located in the basal layer of the VE. Type II cells, about 37% had a single apical process that projected toward the lumen, ending at the epithelial surface. Type III cells sent a prominent process mainly toward the basement membrane, and occupied about 19% of the total cells observed. In the lamina propria, a considerable number of PGP-ir cells was observed. Some of them were present in nerve fiber bundles and contained processes parallel to the bundles. In addition, PGP-ir nerve fiber bundles and cells associated with them were even present in the portion of the nasal septal mucosa that was very close to the brain. The present results strongly suggested that the VE in human fetuses at mid-gestation is a neuroepithelium and that the VE may produce migrating cells toward the brain.

The vomeronasal sensory epithelium of a crotaline snake, Trimeresurus flavoviridis, was shown to consist of a superficial supporting cell layer and an underlying sensory cell layer composed of columns of sensory cells. The supporting cell layer consists of both supporting cells and dendrites of the underlying sensory neurons. The apical regions of sensory cell dendrites contain numerous microtubules, many elongated mitochondria, centrioles, and electron-dense bodies. The dendrites terminate as dendritic knobs from which microvilli project into the vomeronasal lumen. Smooth vesicles are abundant in the dendritic terminals and their vicinity. Supporting cells also bear microvilli, and these cells contain large electron-opaque granules and dense vesicles near their free surfaces. Cytoplasmic extensions of the supporting cells form a meshwork which separates dendrxites from each other in the vicinity of the luminal surface. The meshwork becomes obliterated in the infranuclear region of each supporting cell. Bipolar-shaped sensory cells with lightly stained round nuclei contain the characteristic cell organelles of neurons and are thought to be sensory neurons. These cells are especially characterized by well-developed lamellae of rough endoplasmic reticulum and extensive arrays of smooth endoplasmic reticulum. The perikarya of cells located in the apical region of the cell columns tend to contain larger amounts of smooth endoplasmic reticulum and lipofuscin granules than the perikarya of cells located in lower regions. Undifferentiated cells are found in the basal region of the columns. Satellite cells form the framework of the columns and are also found among neuronal elements.

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 role of nerve growth factor (NGF) in neurotrophic support for the extrinsic innervation of the nasal and oral mucosae was investigated in keratin 14 (K14) - NGF transgenic mice in which NGF was overexpressed in K14-synthesizing cells. K14 immunoreactivity was localized in the epithelial basal cells of the whisker pad skin, the hard palate, the floor of the ventral meatus, and the anterior tongue that are stratified squamous epithelia, and also in basal cells of the vomeronasal, olfactory, and respiratory epithelia that are non-stratified epithelia. In transgenic mice, NGF expression was identified and confined primarily to the basal cells of stratified epithelia. The nasal mucosae including the vomeronasal, olfactory, and respiratory mucosae, and the glands associated with the vomeronasal organ received a greater innervation of protein gene product 9.5-immunoreactive extrinsic fibers in transgenic animals than nontransgenic controls. An increased density of calcitonin gene-related peptide-immunoreactive extrinsic fibers was observed in the nonsensory epithelia of the vomeronasal organ, the olfactory sensory and respiratory epithelia in transgenic animals. Our results indicated that the hyperinnervation of the nasal and oral mucosae by extrinsic neurons is due at least partially to target-derived NGF synthesis and release by K14-expressing basal cells.

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.

Physiologic studies conducted in rats have demonstrated that afferent fibers of the gastric branch of the vagus nerve increase their firing rate with the intragastric administration of the amino acid glutamate, and the increased firing rate is blocked by the depletion of serotonin (5-HT), administration of the blocker for the serotonin type-3 receptor (SR3), or nitric oxide synthase (NOS). To understand glutamate signaling in the gastric mucosa at the cellular level, we have been studying rats as an animal model using anatomic and immunohistochemical procedures. Our results have indicated that 5-HT-immunoreactive (ir) cells are present in the superficial part of the gastric mucosal epithelium and in the base of the fundic glands, whereas immunoreactivity for SR3 is localized in the neck and its vicinity of the fundic glands. Further, NOS1/neuronal NOS-ir cells with a bipolar shape are located in the lamina propria where a dense network of neuronal cells is present. These results suggest that complex cellular events take place during intragastric glutamate signaling.

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.

Immunolocalization of apolipoprotein E (apoE) was investigated in human olfactory mucosa in which olfactory receptor neurons (ORNs) were identified with antiserum to protein gene product (PGP) 9.5. Tissue was obtained at autopsy from 10 nondemented middle-aged or elderly subjects and 9 patients with Alzheimer's disease (AD). Double-labeling immunofluorescence established that apoE immunoreactivity was colocalized in a subpopulation of PGP 9.5-immunoreactive ORNs. The mean number of apoE-immunoreactive ORNs per unit epithelial length in AD patients was about 3.5 times greater than that in nondemented patients, although the mean number of PGP 9.5-immunoreactive ORNs was similar. The apoE-immunopositive Schwann cells in olfactory nerve bundles were the probable source of apoE in the ORNs. The increased numbers of apoE-immunoreactive ORNs in AD patients compared to nondemented subjects demonstrates another manifestation of AD-related neuropathology, in addition to cytoskeletal changes, β-amyloid deposition, and changes in immunoreactivity for other neuroproteins, that parallels changes in neurons in the AD brain.

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 innervation in human taste buds of the foliate and circumvallate papillae was studied immunohistochemically using several neuronal markers in patients with Alzheimer's disease (AD) and their control (ADC) patients. Antisera to protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), tyrosine hydroxylase (TH), dopamine-beta hydroxylase (DbetaH) and calcitonin gene-related peptide (CGRP) were used in immunofluorescence and streptavidin-biotin-peroxidase complex studies. The antiserum to PGP 9.5 stained a greater number of intragemmal nerve fibers in taste buds than that of other antisera. PGP 9.5 immunoreactivity was strictly localized in the nerve fibers, whereas NSE immunoreactivity was observed not only in the nerve fibers, but also in taste bud cells. Intragemmal TH- and DbetaH-immunoreactive nerve fibers were not identified in taste buds. Only a few intragemmal nerve fibers immunoreactive for anti-CGRP antiserum were observe in a small number of taste buds. Furthermore, quantitive analysis in AD and ADC patients demonstrated that the mean number of PGP 9.5-immunoreactive intragemmal nerve fibers in taste buds of the foliate and circumvallate papillae decreased significantly in AD patients. These results indicated that PGP 9.5 is a most suitable molecular marker for the demonstration of the extrinsic innervation in human taste buds, and that the decreased innervation may account partially for the decrement in chemosensory capacity in AD patients.

Effects of overexpression of nerve growth factor (NGF) on mast cell phenotype and numbers were investigated in nasal and oral mucosae and skin of 3- and 6-week-old transgenic mice in which NGF expression in epithelial basal cells was driven by the keratin-14 promoter. Mast cell phenotypes were identified by Alcian blue/safranin and berberine sulfate histochemistry. In the 3-week-old transgenic mice, NGF overexpression had no effect on phenotype except in tongue, where mast cells exhibited mixed or connective tissue phenotypes compared with the mucosal phenotype in the non-transgenic. In 6-week-old transgenic animals, NGF overexpression resulted in the mucosal phenotype in tissues which contained connective tissue or mixed mast cells in non-transgenics. Mast cell hyperplasia occurred at both ages. NGF effects on mast cell phenotype were age-dependent and involve complex microenvironmental interactions.

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.

The olfactory organ of juvenile clearnose skates (Raja eglanteria) was studied with the light and electron microscopes. The organ is ovoid in shape, and its free surface is complicated by the presence of some 20 lamellae. Each lamella has a folded surface lined by a typical neurosensory olfactory epithelium. Bipolar olfactory receptor neurons, ciliated sustentacular cells, and basal cells are the pre-eminent cellular components of the epithelium. Two types of receptor neurons, both bearing microvilli but not cilia, were identified. The type 1 neuron is similar to that previously described in other fishes. The type 2 neuron has a characteristic morphology justifying a separate description. Its dendritic knob is larger than that of type 1, and its microvilli, which are shorter and thicker, are straight and regularly arranged. Tight bundles of filaments provide a skeleton to each microvillus, and these filament bundles reach more than 5 microns down into the dendrite. Type 2 receptor neurons have a well-developed Golgi complex and sparse rough endoplasmic reticulum (rER), whereas type 1 receptor neurons have a less well-developed Golgi complex and a conspicuous system of rER lamellae. The mucous layer on the epithelial surface is provided by the secretion of goblet cells that are situated mostly in the peripheral regions of each lamella. Secretory granules in the sustentacular cells and glands in the lamina propria were not observed.

Expression of the alpha-subunit of the taste-specific G protein alpha-gustducin and the glycolytic enzyme neuron-specific enolase (NSE) was investigated immunohistochemically in human circumvallate and foliate taste papillae. Immunofluorescence for alpha-gustducin was observed in taste cells of both types of papillae and exhibited two patterns of immunofluorescence, plasmalemmal and cytosolic. The plasmalemmal pattern showed intense immunofluorescence localized to the apical region, and was exhibited by most immunoreactive taste cells. In contrast, the cytosolic pattern, observed in one or two immunoreactive cells in a taste bud per section, showed immunofluorescence distributed throughout the cytoplasm. A subpopulation of alpha-gustducin-immunoreactive taste receptor cells, most of which exhibited the cytosolic pattern, also expressed NSE. Optical sectioning, using confocal laser scanning microscopy, demonstrated the highest level of expression of alpha-gustducin in the apical microvillar region of the taste cells in close apposition to the taste pore. These studies indicate conservation of epitopes of alpha-gustducin in humans and rats, and suggest that this G protein is associated with taste transduction in both rats and humans. The patterns of expression of alpha-gustducin, and coexpression with NSE, may correlate with specialized subtypes or developmental stages of taste receptor cells.

Lectinohistochemistry and confocal laser scanning microscopy (CLSM) were utilized to resolve the mucomicrovillar complex (MMC) [1] of the vomeronasal sensory epithelium (VE) into mucoid and sensory components, and to identify glycoconjugates in these components. Six-week-old Sprague-Dawley rats of both sexes were anesthetized deeply with Nembutal and transcardially perfused with Zamboni’s fixative. The vomeronasal organ (VNO), olfactory mucosa (OM), and the septal organ of Masera (SO) were sectioned serially in the transverse plane, using a cryostat. Binding sites for the isolectin Bandeiraea simplicifolia-I B4 (BS-I-B4), which specifically recognizes terminal α-d-galactose (α-Gal) sugar residues [2], were localized on tissue sections with the standard fluorescence technique for fluorescein isothiocyanate (FITC)-BS-I-B4, and the avidinbiotin-peroxidase complex (ABCperoxidase) technique for biotin-conjugated BS-I-B4. Some sections were double-labeled with the antibody against olfactory marker protein (OMP) using the ABC-fluorochrome (Texas Red) technique and with FITC-BS-I-B4 using the fluorescence technique. These sections were analyzed with a confocal laser scanning microscope (MultiProbe 2001; Molecular Dynamics, Sunnyvale, Calif.), using a filter set for simultaneous detection, that is, dual scanning, of the optimal emission spectrum of FITC (peak, 520 nm) and Texas Red (peak, 620 nm).

IMMUNOHISTOCHEMICAL localization of three molecular markers, neuron-specific enolase (NSE) and protein gene product (PGP) 9.5 for neurons and neuroendocrine cells, and olfactory marker protein (OMP) for olfactory receptor neurons (ORNs) was investigated in the vomeronasal epithelium (VNE) of adult humans. NSE- and PGP 9.5-immunoreactive cells were identified in the VNE. ORNs in the olfactory epithelium of approximately age-matched controls were immunoreactive for the three markers. Most NSE-immunoreactive cells in the VNE were bipolar and similar in shape to the NSE- and PGP 9.5-immuno-reactive ORNs. The results indicate that the adult human VNE contains cells expressing two molecular markers characteristic of neurons and that these cells bear a striking morphological similarity to ORNs.

In many terrestrial tetrapodes, a pair of vomeronasal organs (VNOs), which are chemosensory apparatuses, are situated at the base of the nasal septum in the anterior nasal cavity. The purposes of this review are to summarize comparative neuroanatomy and to introduce recent progress in neurobiological studies of the VNO. Five types of VNOs can be identifiable in terms of anatomical organization; snakes possess the most complex one. Sensory cells in the VNO, vomeronasal receptor neurons (VRNs), are located in its neuroepithelium, vomeronassal sensory epithelium. The VRNs retain the characteristic of epithelial cells in that they are born continuously from progenitor cells. They contain two prominent subcellular structures: microvilli and extraordinarily large amounts of smooth endoplasmic reticulum and a few unique glycoconjugates. The VRNs express two types of G-protein -subunits: Gi(alpha2) and Go(alpha) and each of them is coupled with putative pheromone receptors, V1Rs and V2Rs, respectively. Recent physiological and biochemical studies have demonstrated that pheromones depolarize the V1R-Gi(alpha2) and V2R-Go(alpha) VRNs via IP(3)-mediated mechanisms. The VRNs do not show adaptation and are ultrasensitive to putative pheromones. Other than being a chemosensory organ, the VNO and its primordium might play important roles for brain development; hypothalamic neurons that produce gonadotropin-releasing hormone are born in the VNO primordium and a few other neuron-like cells may be born in the VNO primordium and VNO. In human fetuses, anatomical findings strongly suggest that their VNOs contain a neuroepithelium. By contrast, it is unlikely that adult human VNO serves as a chemosensory organ.

We examined the influence of castration on the volumes of sexually dimorphic nuclei, the amygdala pars medialis (Am) and lateralis (Al), and the anterior part of the preoptic nucleus, and on the immunoreactivity of luteinizing hormone-releasing hormone (LH-RH) in brains of Japanese toads captured in spring and autumn. Animals were castrated (GnX) and half were implanted with testosterone (T) (GnX + T) and then killed and dissected after 1 month. Compared with sham-operated (Sham) toads, plasma androgen levels in autumn toads were significantly decreased by the castration, and those in both spring and autumn GnX toads were significantly elevated by the T implantation. The volume of Am in autumn toads was significantly reduced by GnX. Although not significant, changes in the volumes of the other nuclei, except for Al in spring toads, showed the following tendency 30 days after the operation: GnX + T greater than Sham greater than GnX. GnX did not alter LH-RH immunoreactivity in the median eminence. However, dense immunoreactive LH-RH fibers were found in the mesencephalic tegmental region in spring GnX toads but not in the other operation groups in both spring and autumn. LH-RH immunoreactivity was not altered in autumn toads. In spring GnX toads, thumb pads degenerated and evoked release calling was infrequent. These results suggest that (i) the volumes of sexually dimorphic nuclei, especially Am in the autumn toad, are androgen-dependent, and (ii) castration can modulate activity of the extrahypothalamic LH-RH-ergic projection in toad brain.

The anterior part of the preoptic nucleus (APON) in anurans is considered to be a center for mate calling. This locus makes an anatomical complex with the amygdala pars medialis (Am). The nuclear volumes of the Am and APON in the male toad are significantly larger than those in the female. In addition, the nuclear volumes of these loci in the hibernating male are larger than those in the post-breeding animal. These results suggest that the Am-APON complex has an important role in male sexual behavior, and this complex can be activated during the breeding season in the spring.