Continual neurogenesis of vomeronasal neurons in vitro

We developed a culture system of vomeronasal neurons in which continuous degeneration and regeneration of axon bundles were observed. Partially dissociated vomeronasal cells from rat embryonic day 15 were grown in culture and formed a miniature vomeronasal‐like epithelium. We called these structures vomeronasal pockets. They contained both vomeronasal neurons and supporting cells. They formed a spherical structure with a central cavity where microvilli protruded from supporting cells. Mature vomeronasal neurons with well‐developed microvilli were not observed in the vomeronasal pocket. The time period between degeneration of axon bundles and the next was about 2 weeks. When vomeronasal pockets were incubated with 5 µg/mL aphidicolin, an inhibitor of cell division, regeneration of axon bundles was not observed after degeneration. These results suggest that vomeronasal neurons in culture undergo continuous regeneration but do not fully mature. In this culture system, vomeronasal pockets survived for over 1 year. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 226–233, 1999     

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.     

Identification of cytoskeletal markers for the different microvilli and cell types of the rat vomeronasal sensory epithelium

The vomeronasal organ (VNO) of the mammal nose is specialized to detect pheromones. The presumed site of the chemosensory signal transduction of pheromones is the vomeronasal brush border of the VNO sensory epithelium, which has been shown to contain two different sets of microvilli: (i) the tall microvilli of supporting cells and (ii) the short microvilli of the chemoreceptive VNO neurons that branch and intermingle with the basal portions of the longer supporting cell microvilli. A key problem when studying the subcellular distribution of possible VNO signal transduction molecules at the light microscope level is the clear discrimination of immunosignals derived from dendritic microvilli of the VNO neurons and surrounding supporting cell structures. In the present study we therefore looked for cytoskeletal marker proteins, that might help to distinguish at the light microscope level between the two sets of microvilli. By immunostaining we found that the VNO dendritic microvilli can be selectively labelled with antibodies to the calcium-sensitive actin filament-bundling protein villin, whereas supporting cell microvilli contain the actin filament cross-linking protein fimbrin, but not villin. Useful cytoplasmic marker molecules for cellular discrimination were cytokeratin 18 for supporting cells and β-tubulin for dendrites of VNO neurons. A further finding was that the non-sensory epithelium of the rat VNO contains brush cells, a cell type that appears to be involved in certain aspects of chemoreception in the gut. Brush cells or other structures of the vomeronasal brush border did not contain α-gustducin.     

Postnatal development of the vomeronasal epithelium in the rat: an ultrastructural study

Three basic types of cells are distinguished in the rat vomeronasal epithelium at birth: bipolar neurons, supporting cells, and basal cells. Neurons at this time include both immature and differentiated cells. By the end of the first postnatal week, all neurons show morphological signs of maturity in their cytoplasm, including abundant granular and smooth endoplasmic reticulum, neurotubules, dense lamellar bodies, apical centrioles, and tufts of microvilli. During the third week microvilli are more frequently encountered and appear to be longer and more branched. Supporting cells appear well-developed by the second day after birth. During the first ten days of life, supporting cells lose their centrioles and all of the complex associated with ciliary generation in the apical zone. Basal cells appear to be more numerous in newborns than in older animals. Protrusions projecting into the lumen are frequently observed in the epithelium of newborn animals, both on the dendrites of neurons and on supporting cells. After the third week, such protrusions are only observed in the transitional zone between the sensory and the non-sensory epithelia of the vomeronasal tubes. In this transitional zone, a fourth cell type showing apical protrusions with microvilli differentiates. Cytoplasm in this type resembles that of neighboring ciliated cells but has no cilia or centrioles. These transitional cells are considered to be cells in an intermediate state of differentiation, between that of the differentiated neurons and supporting cells of the sensory epithelium and that of the predominate ciliated cells of the non-sensory epithelium. The results suggest that by the end of the third week the vomeronasal epithelium is morphologically mature.     

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.     

Immunocytochemical study of G(i)2alpha and G(o)alpha on the epithelium surface of the rat vomeronasal organ

To investigate in detail the distribution of G protein subtypes G(i)2alpha and G(o)alpha along the surface of the vomeronasal epithelium, we used double labeling immunocytochemical methods and electron microscopy. We examined the immunoreactivity of these surface structures with antibodies against G(i)2alpha and G(o)alpha. G(i)2alpha- and G(o)alpha-positive cells were observed at the epithelial surface and were evenly distributed. Electron microscopy revealed that strong immunoreactivities to both antibodies were observed on the microvilli and knob-like surface structures of receptor cells. No immunoreactivity was found on the microvilli or surface membranes of supporting cells. This expression pattern is similar to that reported for putative pheromone receptors. These data confirm that there are two distinct classes of vomeronasal receptor cells expressed at the surface of the epithelium. These two classes of receptors correspond to the same G(i)2alpha- and G(o)alpha-positive cells distributed in cell body layers of the epithelium and in the axon terminals in the accessory olfactory bulb.     

Differential binding patterns of three antibodies (VOBM1, VOBM2, and VOM2) in the rat vomeronasal organ and accessory olfactory bulb

Immunohistochemical properties of monoclonal antibodies raised against the rat vomeronasal epithelium were examined in adult rats. Three monoclonal antibodies, VOBM1, VOBM2, and VOM2, reacted specifically to the luminal surface of the sensory epithelium of the vomeronasal organ. In addition, the reactivities of VOBM1 and VOBM2 were detected in the vomeronasal nerve layer and the glomerular layer of the accessory olfactory bulb. Electron-microscopic study revealed differential patterns of the immunoreactivity of the three antibodies to the microvilli of vomeronasal sensory epithelium. VOBM1 immunoreactivity was found on the microvilli of the supporting cells, whereas VOBM2 immunoreactivity was found on those of the sensory cells. VOM2 immunoreactivity was observed on the microvilli of both the sensory and supporting cells. These results suggest that the three antibodies recognize different antigens on the vomeronasal sensory epithelium. In particular, VOBM2 antibody appears to react to an antigen specific to the microvilli of the vomeronasal sensory cells.     

Localization of G protein alpha subunits and morphology of receptor neurons in olfactory and vomeronasal epithelia in Reeve's turtle, Geoclemys reevesii

Most vertebrates have two nasal epithelia: the olfactory epithelium (OE) and the vomeronasal epithelium (VNE). The apical surfaces of OE and VNE are covered with cilia and microvilli, respectively. In rodents, signal transduction pathways involve G alpha olf and G alpha i2/G alpha o in OE and VNE, respectively. Reeve's turtles (Geoclemys reevesii) live in a semiaquatic environment. The aim of this study was to investigate the localization of G proteins and the morphological characteristics of OE and VNE in Reeve's turtle. In-situ hybridization analysis revealed that both G alpha olf and G alpha o are expressed in olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs). Immunocytochemistry of G alpha olf/s and G alpha o revealed that these two G proteins were located at the apical surface, cell bodies, and axon bundles in ORNs and VRNs. Electron microscopic analysis revealed that ORNs had both cilia and microvilli on the apical surface of the same neuron, whereas VRNs had only microvilli. Moreover G alpha olf/s was located on only the cilia of OE, whereas G alpha o was not located on cilia but on microvilli. Both G alpha olf/s and G alpha o were located on microvilli of VNE. These results imply that, in Reeve's turtle, both G alpha olf/s and G alpha o function as signal transduction molecules for chemoreception in ORNs and VRNs.     

The vomeronasal cavity in adult humans

We observed the surface of the anterior part of the nasal septum of living subjects using an endoscope. In approximately 13% of 1842 patients without pathology of the septum, the vomeronasal pit was clearly observed on each side of the septum, and in 26% it was observed only on one side. The remaining observations indicated either the presence of putative pits or no visible evidence of a pit. However, repetitive observations on 764 subjects depicted changes over time, from nothing visible to well-defined pits and vice versa. Based on 130 subjects observed at least four times, we estimate that approximately 73% of the population exhibits at least one clearly defined pit on some days. By computer tomography, the vomeronasal cavities were located at the base of the most anterior part of the nasal septum. Histological studies indicated that the vomeronasal cavities consisted of a pit generally connected to a duct extending in a posterior direction under the nasal mucosa. Many glands were present around the duct, which contained mucus. There was no sign of the pumping elements found in other mammalian species. Most cells in the vomeronasal epithelium expressed keratin, a protein not expressed by olfactory neurons. Vomeronasal epithelial cells were not stained by an antibody against the olfactory marker protein, a protein expressed in vomeronasal receptor neurons of other mammals. Moreover, an antibody against protein S100, expressed in Schwann cells, failed to reveal the existence of vomeronasal nerve bundles that would indicate a neural connection with the brain. Positive staining was obtained with the same antibodies on specimens of human olfactory epithelium. The lack of neurons and vomeronasal nerve bundles, together with the results of other studies, suggests that the vomeronasal epithelium, unlike in other mammals, is not a sensory organ in adult humans.     

Ultrastructural localization of α-galactose-containing glycoconjugates in the rat vomeronasal organ

Binding sites of Griffonia simplicifolia I-B₄ isolectin (GS-I-B₄), which recognizes terminal α-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 α-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 α-galactose-containing glycoconjugates, compared with those in sustentacular microvilli. Further, biochemical analyses demonstrated several vomeronasal organ-specific glycoproteins with terminal α-galactose.     

Engulfment of Axon Debris by Microglia Requires p38 MAPK Activity

The clearance of debris after injuries to the nervous system is a critical step for restoration of the injured neural network. Microglia are thought to be involved in elimination of degenerating neurons and axons in the central nervous system (CNS), presumably restoring a favorable environment after CNS injuries. However, the mechanism underlying debris clearance remains elusive. Here, we establish an in vitro assay system to estimate phagocytosis of axon debris. We employed a Wallerian degeneration model by cutting axons of the cortical explants. The cortical explants were co-cultured with primary microglia or the MG5 microglial cell line. The cortical neurites were then transected. MG5 cells efficiently phagocytosed the debris, whereas primary microglia showed phagocytic activity only when they were activated by lipopolysaccharide or interferon-β. When MG5 cells or primary microglia were co-cultured with degenerated axons, p38 mitogen-activated protein kinase (MAPK) was activated in these cells. Engulfment of axon debris was blocked by the p38 MAPK inhibitor SB203580, indicating that p38 MAPK is required for phagocytic activity. Receptors that recognize dying cells appeared not to be involved in the process of phagocytosis of the axon debris. In addition, the axons undergoing Wallerian degeneration did not release lactate dehydrogenase, suggesting that degeneration of the severed axons and apoptosis may represent two distinct self-destruction programs. We observed regrowth of the severed neurites after axon debris was removed. This finding suggests that axon debris, in addition to myelin debris, is an inhibitory factor for axon regeneration.Axon degeneration is an active, tightly controlled, and versatile process of axon segment self-destruction. The lesion-induced degeneration process was first described by Waller (1) and has since been known as Wallerian degeneration (2, 3). This degeneration involves rapid blebbing and fragmentation of an entire axonal stretch into short segments, which are then removed by locally activated phagocytic cells. Phagocytic removal of damaged axons and their myelin sheaths distal to the injury is important for creating a favorable environment for axonal regeneration in the nervous system. Although the debris of degenerated axons and myelin is cleared by phagocytes in the peripheral nervous system (PNS), the debris is removed very slowly in the central nervous system (CNS)³ (4, 5). This is considered to be one of the obstacles for regeneration of the injured axons in the CNS.Apoptotic neurons are also engulfed by activated phagocytic cells. Apoptosis is very well documented in the CNS where a significant proportion of neurons undergo programmed cell death (6). To prevent the diffusion of damaging degradation products into surrounding tissues, dying neurons are phagocytosed. In the brain, apoptotic cells are engulfed mainly by the resident population of phagocytes known as microglia. Microglia are generally considered to be immune cells of the CNS (7). They respond to any kind of pathology with a reaction termed “microglial activation.” After injuries to the CNS, microglia react within a few hours with a migratory response toward the lesion site.Although insight into the mechanism of phagocytosis of dying cells by microglia has improved, little is known about the mechanism of clearance of degenerated axons and myelin debris by microglia after axonal injury in the CNS. Interestingly, the axons undergoing Wallerian degeneration do not seem to possess detectable activation of the caspase family (8), suggesting that Wallerian degeneration and apoptosis may represent two distinct self-destruction programs. Thus, the mechanism of microglial phagocytosis of dying cells might be different from that of axon/myelin debris. We aimed to elucidate the mechanism of debris clearance by microglia after an axonal injury. We established an in vitro assay system to estimate phagocytosis of degenerated axon debris. We found that p38 mitogen-activated protein kinase (MAPK) was critical for the phagocytic activity of microglia. Treatment with lipopolysaccharide (LPS) or interferon-β (IFN-β) was necessary for the primary microglia to become phagocytic. In addition, clearance of degenerated axon debris allowed axonal growth from the severed neurites, suggesting that removal of the axon debris provides a favorable environment for axonal regeneration.     

Light and electron microscopic observations on the normal structure of the vomeronasal organ of garter snakes

The vomeronasal epithelium of adult garter snakes (Thamnophis sirtalis and T. radix) was studied by light and electron microscopy. The sensory epithelium is extraordinarily thick, consisting of a supporting cell layer, a bipolar cell layer, and an undifferentiated cell layer. The supporting cell layer is situated along the luminal surface and includes supporting cells and the peripheral processes (dendrites) of bipolar neurons. The luminal surfaces of both supporting cells and bipolar neurons are covered with microvilli. Specializations of membrane junctions are always observed between adjacent cells in the subluminal region. Below the supporting cell layer, the epithelium is characterized by a columnar organization. Each column contains a population of bipolar neurons and undifferentiated cells. These cells are isolated from the underlying vascular and pigmented connective tissue by the presence of a thin sheath of satellite cells and a basal lamina. Heterogeneity of cell morphology occurs within each cell column. Generative and undifferentiated cells occupy the basal regions and mature neurons occupy the apical regions. Transitional changes in cell morphology occur within the depth of each cell column. These observations suggest that the vomeronasal cell column is the structural unit of the organ and may represent the dynamic unit for cell replacement as well. A sequential process of cell proliferation, neuronal differentiation, and maturation appears to occur in the epithelium despite the adult state of the animal.     

The cell coat of the olfactory epithelium proper and vomeronasal neuroepithelium of the rat as revealed by means of the Ruthenium-red reaction

Summary The apical cell coat of the olfactory epithelium proper and the vomeronasal neuroepithelium of the rat was investigated electronmicroscopically by means of the Ruthenium-red reaction. In the olfactory epithelium proper, the cilia of receptor cells and microvilli of supporting cells possess a cell coat measuring approximately 10 nm in thickness. In the vomeronasal neuroepithelium, the apical cell coat is thicker than in the olfactory epithelium proper. On microvilli of vomeronasal receptor cells the cell coat varies in thickness from 15 to 20 nm, and on microvilli of supporting cells it measures approximately 75 nm. The functional implications of these findings are discussed.A portion of this study was presented at the 6^th European Anatomical Congress in Hamburg. This publication is dedicated to Prof. E. KlikaSupported by the Deutsche Forschungsgemeinschaft (Br 358/5-1).     

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons

Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca²⁺ concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca²⁺ in isolated mouse vomeronasal sensory neurons in the absence of intracellular K⁺. In whole-cell recordings, the average current in 1.5 µM Ca²⁺ and symmetrical Cl⁻ was −382 pA at −100 mV. Ion substitution experiments and partial blockade by commonly used Cl⁻ channel blockers indicated that Ca²⁺ activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca²⁺-activated Cl⁻ channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca²⁺-activated Cl⁻ channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre–loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca²⁺-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca²⁺-activated Cl⁻ currents in mouse vomeronasal sensory neurons.     

Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis

BACKGROUND: Axon pruning is involved in establishment and maintenance of functional neural circuits. During metamorphosis of Drosophila, selective pruning of larval axons is developmentally regulated by ecdysone and caused by local axon degeneration. Previous studies have revealed intrinsic molecular and cellular mechanisms that trigger this pruning process, but how pruning is accomplished remains essentially unknown. RESULTS: Detailed analysis of morphological changes in the axon branches of Drosophila mushroom body (MB) neurons revealed that during early pupal stages, clusters of neighboring varicosities, each of which belongs to different axons, disappear simultaneously shortly before the onset of local axon degeneration. At this stage, bundles of axon branches are infiltrated by the processes of surrounding glia. These processes engulf clusters of varicosities and accumulate intracellular degradative compartments. Selective inhibition of cellular functions, including endocytosis, in glial cells via the temperature-sensitive allele of shibire both suppresses glial infiltration and varicosity elimination and induces a severe delay in axon pruning. Selective inhibition of ecdysone receptors in the MB neurons severely suppressed not only axon pruning but also the infiltration and engulfing action of the surrounding glia. CONCLUSIONS: These findings strongly suggest that glial cells are extrinsically activated by ecdysone-stimulated MB neurons. These glial cells infiltrate the mass of axon branches to eliminate varicosities and break down axon branches actively rather than just scavenging already-degraded debris. We therefore propose that neuron-glia interaction is essential for the precisely coordinated axon-pruning process during Drosophila metamorphosis.     

Cell dynamics in the olfactory mucosa

By means of ultrastructural and autoradiographic observations from the olfactory mucosa of frog, it has been shown that olfactory receptor neurons as well as supporting cells are continuously replaced during the adult life of the animal. The severing of the olfactory nerve in adult frogs results in rapid degeneration of all mature olfactory neurons. An increased mitotic activity of the basal cells accompanies the degeneration of the mature neurons and precedes the regeneration of new neurons. The capability of these newly formed neurons to re-establish their connections in the olfactory bulb has been ascertained and the modalities of the process will be dealt with in a further report.     

A long-term culture system for olfactory explants with intrinsically fluorescent cell populations

As a prerequisite for exploring the mechanisms which lead to the formation and maintenance of the precise wiring patterns in the olfactory system, organotypic cultures of olfactory tissue from transgenic mice expressing green fluorescent protein (GFP) under control of the olfactory marker protein promotor have been established. Tissue specimen from embryonic stage 14 were explanted and kept in culture for >1 week. Within the explants, numerous GFP-fluorescent olfactory sensory neurons assembled in an epithelial-like manner during this period. Under optimized culture conditions, strongly GFP-positive axons extended from these explants, fasciculated and formed bundles. When co-cultured with explants from the olfactory bulb, distinct axon populations were attracted by the target tissue; the fluorescent axon bundles invaded the bulbular explants and formed conglomerates at distinct spots. Explants from transgenic mice expressing GFP under control of a given olfactory receptor gene (mOR37A) also comprised labeled neurons that extended intensely fluorescent axonal processes, which all seemed to grow in a common fascicle. The results demonstrate that GFP-labeled olfactory sensory neurons differentiate in the established organotypic cultures, which thus appear to be a useful tool to monitor and to manipulate the processes underlying the axonal wiring between the olfactory epithelium and bulb.     

The characteristics of the electrovomeronasogram: its loss following vomeronasal axotomy in the garter snake

Electrovomeronasogram (EVG) recordings were made from adult garter snakes, Thamnophis sirtalis. Stimulation of vomeronasal epithelium with a stimulus prepared from prey, earthworm electric shock secretion (ESS), evoked EVG response in a dose-dependent manner. The magnitude of the EVG response to ESS was remarkably larger than n-amyl acetate and glutamate, which elicited insignificant responses, supporting the idea that the vomeronasal system is differentially sensitive to liquid delivery of biologically significant chemical stimuli. Fourteen days following vomeronasal axotomy, the magnitudes of the EVG responses of animals which received bilateral axotomy without cauterization or with cauterization was -0.19+/-0.07 mV or -0.05+/-0.02 mV respectively, compared with the normal EVG response of -0.41+/-0.10 mV. The epithelia of animals which received bilateral axotomy without cauterization exhibited remarkable degeneration of the bipolar neurons. Maximal depletion of bipolar neurons occurred in the epithelia denervated with cauterization, though the difference between cell densities in vomeronasal neuron layers in these epithelia was not statistically significant. The present results clearly indicate that the fewer neurons the epithelium contains, the smaller EVG response it generates, suggesting that the receptor neurons are the primary origin of EVG responses.      

Anatomy,histology, and histochemistry of the olfactory organ of the Korean shuttles mudskipper Periophthalmus modestus

The Korean shuttles mudskipper Periophthalmus modestus has paired olfactory organs on its snout, consisting of anterior and posterior nostrils, a single olfactory canal with sensory and nonsensory epithelia, and a single accessory nasal sac. Its sensory epithelium consists of numerous islets forming a pseudostratified layer and contains various cells: olfactory receptor neurons, supporting cells, basal cells, lymphatic cells (LCs), and axon bundles. The sensory epithelium is a stratified squamous layer comprising stratified epithelial cells, mucous cells (MCs) with glycogen, flattened cells (FCs), LCs, and unidentified cells. Specific structures are as follows: (a) a tubular anterior nostril projecting outward, (b) a slit posterior nostril, (c) an elongated olfactory canal, (d) an ethmoidal accessory nasal sac, (e) axon bundles found only in the basal layer of the sensory epithelium, (f) FCs only at the top of the nonsensory epithelium, and (g) glycogen-containing MCs. Such structures seem to be unique in that they have not been observed in most teleost fishes spending their whole life in water.     

Vomeronasal receptors in Turtles

Summary The functional similarities observed with electrophysiological techniques between olfactory and vomeronasal receptors allow speculation that morphological details essential to the common function should be observed in both cases. Both mucosae have primary receptors within the epithelium which are surrounded, but not completely isolated, by so-called supporting cells. These last secrete a granular product. In both epithelia receptor cells contact each other at the axonal, perikaryal, dendritic and junctional complex levels. The axons of the two types of receptors are unmyelinated and their diameter ranges from 0.1 to 0.4 micron. The most interesting difference between the two types of receptors lies at the level of their exposed endings. The olfactory vesicle, as it is classically represented in olfactory receptors and is common in those of turtles in the form of a ball-like protrusion above the epithelial surface, is usually missing in the vomeronasal receptors. These have a tapering cone-shaped irregular projection always complicated by a set of branched microvilli. They do, furthermore, consistently lack cilia. This observation is in agreement with recent TEM observations. The assumption that cilia are essential in the mechanism of olfactory transduction is discussed on the basis of these anatomical findings.