Nobuaki Nakamuta

Histological and Ultrastructural Characteristics of the Primordial Vomeronasal Organ in Lungfish

Many vertebrates have two anatomically distinct olfactory organs—the olfactory epithelium and the vomeronasal organ—to detect chemicals such as general odorants and pheromones in their environment. The vomeronasal organ is not present in fish but is present in vertebrates of a higher order than amphibians. Among all extant fishes, the lungfish is considered to be genetically and phylogenetically closest to tetrapods. In this study, we examined the olfactory organs of African lungfish, Protopterus annectens, by lectin histochemistry, immunohistochemistry, and transmission electron microscopy. Two types of sensory epithelia were identified in the olfactory organ—the olfactory epithelium covering the surface of lamellae and the sensory epithelium lining the recesses both at the base of lamellae and in the wall of the nasal sac—and designated here as the lamellar olfactory epithelium and the recess epithelium, respectively. Based on analysis of G‐protein expression and ultrastructure, the lamellar olfactory epithelium resembled the olfactory epithelium of ordinary teleosts and the recess epithelium resembled the vomeronasal organ of tetrapods. Furthermore, lectin histochemistry demonstrated that the axons from the recess epithelium converge and project to the ventrolateral part of the olfactory bulb, suggesting that lungfish possess a region homologous to the accessory olfactory bulb of tetrapods. Based on these results, it seems appropriate to refer to the recess epithelium as “a primordium of the vomeronasal organ.” This study may provide important clues to elucidate how the vomeronasal organ emerged during the evolution of vertebrates. Anat Rec, 2012.

Lectin histochemical studies on the olfactory epithelium and vomeronasal organ in the Japanese striped snake, Elaphe quadrivirgata.

The olfactory epithelium and the vomeronasal organ of the Japanese striped snake were examined by lectin histochemistry. Of the 21 lectins used in the study, all lectins except succinylated‐wheat germ agglutinin (s‐WGA) showed similar binding patterns in the vomeronasal receptor cells and the olfactory receptor cells with varying intensities. The binding patterns of s‐WGA varied among individuals in the vomeronasal and olfactory receptor cells, respectively. Four lectins, Bandeiraea simplicifolia lectin‐II (BSL‐II), Dolichos biflorus agglutinin (DBA), Sophora japonica agglutinin (SJA), and Erythrina cristagalli lectin (ECL) stained secretory granules and the organelles in the olfactory supporting cells and did not stain them in the vomeronasal supporting cells. These results suggest that the glycoconjugate moieties are similar in the vomeronasal and olfactory receptor cells of the Japanese striped snake. J. Morphol., 2010.

Differences in respiratory changes and Fos expression in the ventrolateral medulla of rats exposed to hypoxia, hypercapnia, and hypercapnic hypoxia.

Respiratory responses to hypoxia and/or hypercapnia, and their relationship to neural activity in the ventrolateral medulla (VLM), which includes the respiratory center, have not yet been elucidated in detail. We herein examined respiratory responses during exposure of 10% O2 (hypoxia), 10% CO2 (hypercapnia), and 10% O2-10% CO2 (hypercapnic hypoxia) using plethysmography. In addition to recording respiration, Fos expressions were examined in the VLM of the rat exposed to each gas to analyze neural activity. Respiratory frequency was increased in rats exposed to hypoxia, and Fos-positive neurons were observed in the caudal VLM (cVLM) and medial VLM (mVLM). Tidal volume was increased in rats exposed to hypercapnia, and Fos-positive neurons were observed in the rostral VLM (rVLM) includes the retrotrapezoid nucleus (RTN) and mVLM. Tidal volume was enhanced in rats exposed to hypercapnic hypoxia, similar to that in hypercapnia-exposed rats, and Fos-positive neurons were observed in the entire region of the VLM. In the mVLM and cVLM, double immunofluorescence showed Fos-immunoreactive nerve cells were also immunoreactive to dopamine β-hydroxylase, the marker for A1/C1 catecholaminergic neuron. These results suggested that hypoxia and hypercapnia modulated rhythmogenic microcircuits in the mVLM via A1/C1 neurons and the RTN, respectively.

Distinct axonal projections from two types of olfactory receptor neurons in the middle chamber epithelium of Xenopus laevis.

Most vertebrates have two olfactory organs, the olfactory epithelium (OE) and the vomeronasal organ. African clawed frog, Xenopus laevis, which spends their entire life in water, have three types of olfactory sensory epithelia: the OE, the middle chamber epithelium (MCE) and the vomeronasal epithelium (VNE). The axons from these epithelia project to the dorsal part of the main olfactory bulb (d-MOB), the ventral part of the MOB (v-MOB) and the accessory olfactory bulb, respectively. In the MCE, which is thought to function in water, two types of receptor neurons (RNs) are intermingled and express one of two types of G-proteins, Golf and Go, respectively. However, axonal projections from these RNs to the v-MOB are not fully understood. In this study, we examined the expression of G-proteins by immunohistochemistry to reveal the projection pattern of olfactory RNs of Xenopus laevis, especially those in the MCE. The somata of Golf- and Go-positive RNs were separately situated in the upper and lower layers of the MCE. The former were equipped with cilia and the latter with microvilli on their apical surface. These RNs are suggested to project to the rostromedial and the caudolateral regions of the v-MOB, respectively. Such segregation patterns observed in the MCE and v-MOB are also present in the OE and olfactory bulbs of most bony fish. Thus, Xenopus laevis is a very interesting model to understand the evolution of vertebrate olfactory systems because they have a primitive, fish-type olfactory system in addition to the mammalian-type olfactory system.

Developmental Changes in Lectin‐Binding Patterns of Three Nasal Sensory Epithelia in Xenopus laevis

The nasal cavity of adult Xenopus laevis (X. laevis) is composed of a series of three compartments: principal, middle, and inferior chambers. The principal chamber is lined with olfactory epithelium (OE), middle chamber with middle chamber epithelium (MCE), and inferior chamber with vomeronasal epithelium (VNE). In the present study, we examined developmental changes of lectin‐binding patterns of the OE, MCE, and VNE by the use of four biotinylated lectins; DSL, DBA, PNA, and UEA‐I. From Stage 59, just after the beginning of metamorphosis, the stainings of the free border for DBA and UEA‐I were decreased in the OE and MCE, respectively, but the stainings of secretory granules (SGs) in the OE became intense. From Stage 63, sensory cells positive for DSL were increased in these three epithelia, and positive stainings for UEA‐I and DBA increased in the SGs and Jacobsons glands (JGs), respectively. In addition, from 3 months after the end of metamorphosis, the stainings of sensory cells for PNA, DBA, and DSL changed in the OE, MCE, and VNE, respectively, and those of the SGs, Bowmans glands, and JGs also changed for several lectins. The present results showed that glycoconjugates expressed in three epithelia and their associated glands changed during and after the end of metamorphosis. These findings may indicate that the functional maturation of each epithelium depends not only on the maturation of sensory cells, but also on the maturation of the SGs in supporting cells of the OE and their associated glands after the end of metamorphosis. Anat Rec, 2011.

Lectin Histochemical Analysis of the Olfactory Bulbs in the Barfin Flounder (Verasper moseri)

Several lines of evidence have shown that the olfactory system of the fish contains the main and accessory olfactory systems. However, morphological data indicate that the accessory olfactory bulb, the primary centre for the accessory olfactory system, will not differentiate in the fish. Therefore, the fish olfactory bulb is supposed to engage in both main and accessory olfactory systems. To examine this possibility, we investigated the olfactory bulb of the barfin flounder (Verasper moseri) by histochemical examination using lectins. The olfactory bulb of the barfin flounder showed a laminar structure with four layers, and diffuse glomerular architecture was observed in the glomerular layer. Based on the expression patterns of sugar residues, the glomerular layer of the barfin olfactory bulb was largely divided into three portions. Heterogeneity in the lectin‐binding pattern among olfactory glomeruli was clearly demonstrated by the fluorescent double‐lectin staining. The results of this study suggest that the fish olfactory bulb contains both regions equivalent to the main and accessory olfactory bulbs, and they are subdivided into small subsets with different functions.

Morphology of P2X3-immunoreactive nerve endings in the rat laryngeal mucosa.

The morphological characteristics of P2X3-immunoreactive nerve endings in the laryngeal mucosa were herein examined using immunohistochemistry with confocal laser microscopy. Ramified intraepithelial nerve endings immunoreactive to P2X3 were distributed in the epiglottis and arytenoid region. The axon terminals of P2X3-immunoreactive ramified endings were beaded or flat in shape. These endings were also immunoreactive to P2X2 and not identical to the nerve endings immunoreactive to Na+-K+-ATPase α3-subunit, substance P (SP), and calcitonin gene-related peptide (CGRP). P2X3-immunoreactive axon terminals were also immunoreactive to vGLUT1, vGLUT2, and vGLUT3. In addition to ramified endings, P2X3-immunoreactive nerve endings were associated with α-gustducin-immunoreactive solitary chemosensory cells and/or SNAP25-immunoreactive neuroendocrine cells. Furthermore, P2X3-immunoreactive nerve endings were also observed in the taste bud-like chemosensory cell clusters of the stratified squamous epithelium covering epiglottic and arytenoid cartilage. The P2X3-immunoreactive nerve endings that associated with sensory and/or endocrine cells and chemosensory cell clusters were also immunoreactive to P2X2, vGLUT1, vGLUT2, and vGLUT3, but not to SP or CGRP. In conclusion, P2X3-immunoreactive nerve endings may be classified into two types, i.e., intraepithelial ramified nerve endings and nerve endings associated with chemosensory cells and neuroendocrine cells.

Immunohistochemical studies for the neuronal elements in the vomeronasal organ of the one-humped camel.

The neuronal elements of the vomeronasal organ (VNO) of camel were investigated immunohistochemically. PGP 9.5 labeled the receptor cells in the vomeronasal sensory epithelium, but not the supporting or basal cells. OMP stained some receptor cells, but no immunoreactive signals for OMP were detected in the non-sensory epithelium. PLCβ2 labeled scattered cells in the sensory epithelium and a larger number of cells in the non-sensory epithelium. Double labeling immunohistochemistry revealed that the PLCβ2-positive cells were surrounded by substance P-positive nerve fibers. Collectively, these data suggest that the camel VNO bears, in addition to the mature vomeronasal receptor cells, trigeminally-innervated solitary chemosensory cells which are expected to play a substantial role in the control of stimulus access to the VNO.

Histological and Lectin Histochemical Studies on the Olfactory and Respiratory Mucosae of the Sheep

ABSTRACT The olfactory and respiratory mucosae of the Corriedale sheep were examined using lectin histochemistry in order to clarify the histochemical and glycohistochemical differences between these two tissues. The olfactory epithelium was stained with 13 lectins out of 21 lectins examined, while the respiratory epithelium was positive to 16 lectins. The free border of both of the olfactory and respiratory epithelia was stained with 12 lectins: Wheat germ agglutinin (WGA), succinylated-wheat germ agglutinin (s-WGA), Lycopersicon esculentum lectin (LEL), Solanum tuberosum lectin (STL), Datura stramonium lectin (DSL), Soybean agglutinin (SBA), Bandeiraea simplicifolia lectin-I (BSL-I), Ricinus communis agglutinin-I (RCA-120), Erythrina cristagalli lectin (ECL), Concanavalin A (Con A), Phaseolus vulgaris agglutinin-E (PHA-E) and Phaseolus vulgaris agglutinin-L (PHA-L). The associated glands of the olfactory mucosa, Bowman’s glands, were stained with 13 lectins. While both the goblet cells and mucous nasal glands were stained with 8 lectins; five of them (WGA, s-WGA, STL, Vicia villosa agglutinin (VVA) and ECL) were mutually positive among the Bowman’s glands, mucous nasal glands and the goblet cells. These findings indicate that the glycohistochemical characteristics of the free borders of both olfactory and respiratory epithelia are similar to each other, suggesting that secretions from the Bowman’s glands and those of the goblet cells and mucous nasal glands are partially exchanged between the surface of two epithelia to contribute the functions of the respiratory epithelium and the olfactory receptor cells, respectively.

Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat

The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO2 and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body.