Shigeru Takami

Immunolocalization of Water Channel Aquaporins in the Vomeronasal Organ of the Rat: Expression of AQP4 in Neuronal Sensory Cells

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.

Immunoelectron microscopic analysis of the distribution of tyrosine kinase receptor B in olfactory axons

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.

Presence of Sex Steroid‐Metabolizing Enzymes in the Olfactory Mucosa of Rats

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 Bowmans 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 Bowmans 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.

Structure and function of the olfactory system: Overview

The main olfactory and vomeronasal systems are major nasal chemosensory systems in mammals (Breer et al., 2006). The olfactory system is composed of a sensory epithelium, termed ‘olfactory epithelium’ (OE), its primary brain center, olfactory bulb (OB), and higher brain centers located in the cerebrum and diencephalon (Fig. 1, Graziadei, 1977, 1990; Farbman 1992). The most remarkable characteristics of the olfactory system are as follows. Olfactory receptor cells (ORC) are bipolar neurons that directly make synapses on OB neurons, but are replaced continuously within the OE throughout an individual’s lifetime. Following mitosis of a basal cell located in the bottom of the OE, a progenitor cell differentiates to an immature ORC, a mature ORC, and finally dies (Graziadei, 1977, 1990; Farbman, 1992). Each ORC sends a single dendrite to the surface of the OE to make a bulbar ending, called an olfactory vesicle or olfactory knob, from which sensory cilia project (Farbman, 1992). Thus, even the OE of mature animals contains mature and immature ORC, and mitotic cells (Fig. 2). Major findings derived from anatomical studies conducted in late 1960s–1980s included the continuous turnover of ORC in the OE, and elucidation of cellular and subcellular structures in the olfactory system. Using radio-autographic techniques, Pasquale P. C. Graziadei and colleagues in Florida State University proved that ORC were derived from basal cells located in the OE and that these cells, even in adult vertebrates, were capable of differentiating to mature ORC (for reviews, see Graziadei, 1977, 1990). The development of electron microscopy and histochemistry techniques greatly contributed to the description of subcellular structures and characterization of constituent cells in the olfactory system (Farbman, 1992; Schwob, 2002). Following the discovery of G-protein-coupled odorant receptors by Linda Buck and Richard Axel (Buck & Axel, 1991), who received the Nobel Prize in Physiology or Medicine 2004, a considerable number of anatomical studies in the mammalian olfactory system were carried out by scientists with a strong molecular biology background. Examples of these major findings are: single ORC expresses only one of the aforementioned odorant receptors, a given odorant receptor is randomly dispersed within a broad but circumscribed zone of the OE, and axons from ORC expressing a given odorant receptor converge to a single locus of the OB (Buck, 2004; Bargmann, 2006; Breer et al., 2006). Although very intensive studies in mainly the OE