Improvement of the vomeronasal organ ablation in the rat

The vomeronasal organ (VNO) in most mammalian species consists of a bilaterally paired tubular structure lying on either side at the base of the nasal septum. Functionally, the VNO receives chemosensory informations and conveys it to its parts of the hypothalamus thought to be important in the regulation of reproductive physiology and behavioral functions. Here the authors report an improvement of the VNO ablation in the rat. With the exception of restraint of a rat, this operation method is fundamentally similar to those reported by Saito and Mennella (1986). The rat was anesthetized and placed supine on a home-made operating board with some accessories, and its extremities and head were fixed. It should be emphasized that the restraint of its lower incisor and tongue to keep the mouth open was performed with home-made restraint devices. The above method enabled us to remove the VNO easily. In addition, it could be useful in this field of oral surgery in small animals.     

Cyclic adenosine monophosphate signaling in the rat vomeronasal organ: role of an adenylyl cyclase type VI

The present study indicates that male rat urinary components in female rat vomeronasal organ microvillar preparations not only induce a rapid and transient IP(3) signal, but in addition, the level of cAMP decreases with a delayed and sustained time course. This decrease seems to be a consequence of the preceding activation of the phosphoinositol pathway rather than the result of an enhanced phosphodiesterase activity or an inhibition of adenylyl cyclase (AC) via Galpha(i) or Galpha(o). This notion is supported by the finding that activation of the endogenous protein kinase C suppresses basal as well as forskolin-induced cAMP formation. Furthermore, it was observed that elevated levels of calcium inhibit cAMP formation in rat VNO microvillar preparations. These properties of cAMP signaling in the VNO of rats may be mediated by a calcium- and protein kinase C-inhibited AC VI subtype, which is localized in microvillar preparations of the VNO.     

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.     

Type-specific inositol 1,4,5-trisphosphate receptor localization in the vomeronasal organ and its interaction with a transient receptor potential channel,TRPC2

The vomeronasal organ (VNO) is the receptor portion of the accessory olfactory system and transduces chemical cues that identify social hierarchy, reproductive status, conspecifics and prey. Signal transduction in VNO neurons is apparently accomplished via an inositol 1,4,5-trisphosphate (IP3)-activated calcium conductance that includes a different set of G proteins than those identified in vertebrate olfactory sensory neurons. We used immunohistochemical (IHC) and SDS-PAGE/western analysis to localize three IP3 receptors (IP3R) in the rat VNO epithelium. Type-I IP3R expression was weak or absent. Antisera for type-II and -III IP3R recognized appropriate molecular weight proteins by SDS-PAGE, and labeled protein could be abolished by pre-adsorption of the respective antibody with antigenic peptide. In tissue sections, type-II IP3R immunoreactivity was present in the supporting cell zone but not in the sensory cell zone. Type-III IP3R immunoreactivity was present throughout the sensory zone and overlapped that of transient receptor potential channel 2 (TRPC2) in the microvillar layer of sensory epithelium. Co-immunoprecipitation of type-III IP3R and TRPC2 from VNO lysates confirmed the overlapping immunoreactivity patterns. The protein-protein interaction complex between type-III IP3R and TRPC2 could initiate calcium signaling leading to electrical signal production in VNO neurons.     

Expression of candidate pheromone receptor genes in vomeronasal neurons

In mammals, olfactory sensory perception is mediated by two anatomically and functionally distinct organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VON). Pheromones activate the VNO and elicit a characteristic array of innate reproductive and social behaviors, along with dramatic neuroendocrine responses. Recent approaches have provided new insights into the molecular biology of sensory transduction in the VNO. Differential screening of cDNA libraries constructed from single sensory neurons from the rat VNO has led to the isolation of a family of genes which are likely to encode mammalian pheromone receptors. The isolation of these receptors from the VNO might permit the analysis of the molecular events which translate the bindings of pheromones into innate stereotypic behaviors and help to elucidate the logic of pheromone perception in mammals.      

Expression of glycoproteins in the vomeronasal organ reveals a novel spatiotemporal pattern of sensory neurone maturation

The main olfactory and the accessory olfactory systems are both anatomically and functionally distinct chemosensory systems. The primary sensory neurones of the accessory olfactory system are sequestered in the vomeronasal organ (VNO), where they express pheromone receptors, which are unrelated to the odorant receptors expressed in the principal nasal cavity. We have identified a 240 kDa glycoprotein (VNO(240)) that is selectively expressed by sensory neurones in the VNO but not in the main olfactory neuroepithelium of mouse. VNO(240) is first expressed at embryonic day 20.5 by a small subpopulation of sensory neurones residing within the central region of the crescent-shaped VNO. Although VNO(240) was detected in neuronal perikarya at this age, it was not observed in the axons in the accessory olfactory bulb until postnatal day 3.5. This delayed appearance in the accessory olfactory bulb suggests that VNO(240) is involved in the functional maturation of VNO neurones rather than in axon growth and targeting to the bulb. During the first 2 postnatal weeks, the population of neurones expressing VNO(240) spread peripherally, and by adulthood all primary sensory neurones in the VNO appeared to be expressing this molecule. Similar patterns of expression were also observed for NOC-1, a previously characterized glycoform of the neural cell adhesion molecule NCAM. To date, differential expression of VNO-specific molecules has only been reported along the rostrocaudal axis or at different apical-basal levels in the neuroepithelium. This is the first demonstration of a centroperipheral wave of expression of molecules in the VNO. These results indicate that mechanisms controlling the molecular differentiation of VNO neurones must involve spatial cues organised, not only about orthogonal axes, but also about a centroperipheral axis. Moreover, expression about this centroperipheral axis also involves a temporal component because the subpopulation of neurones expressing VNO(240) and NOC-1 increases during postnatal maturation.     

Ontogenetic characteristics of the vomeronasal organ in Saguinus geoffroyi and Leontopithecus rosalia,with comparisons to other primates

It has been suggested that the variability of the primate vomeronasal organ (VNO) may be greater than previously thought, especially among New World monkeys. It is not clear to what extent VNO variation reflects ontogenetic, functional, or phylogenetic differences among primates. The present study investigated VNO anatomy in an ontogenetic series of two genera of callitrichid primates, in order to assess recent attempts to develop VNO character states and to examine the evidence for VNO functionality at different life stages. A sample of six Leontopithecus rosalia, one L. chrysomelas, and six Saguinus geoffroyi was serially sectioned and stained using various methods. Two adult Callithrix jacchus were also sectioned for comparative purposes. The VNO of each primate was examined by light microscopy along its entire rostrocaudal extent. VNOs of the tamarins were described to determine whether they fit into 1 of 3 character states recently attributed to various New World monkeys. At birth, the two species of tamarins differed in the nature of communication between the VNO and nasopalatine duct (NPD). Two of 3 neonatal S. geoffroyi exhibited a fused VNO duct in a more dorsal position (adjacent to the nasal cavity) compared to that of L. rosalia. The VNO duct communicated with the NPD and was patent in neonatal L. rosalia. Both species appeared to have an age-related increase in the amount of sensory epithelium in the VNO. Subadult L. rosalia had caudal regions of the VNO that were exceptionally well-developed, similar to those of strepsirhine primates. Compared to subadults, all adult callitrichids appeared to have more ventral communications of the VNO duct directly into the NPD. Adult S. geoffroyi and L. chrysomelas both had VNO sensory epithelium separated by multiple patches of nonsensory epithelium. This contrasted with the VNOs of C. jacchus, which had a nearly continuous distribution of receptors on all surfaces of the VNO. The findings indicate that tamarins have delayed maturation of the VNO epithelium, and that some species have little or no perinatal function. These results also suggest that ontogenetic changes in craniofacial form may alter the position of the VNO in tamarins. The present study supports the use of at least two character states to categorize the VNO of various callitrichids, but it is suggested that one of these, previously called "reduced sensory epithelium" should be instead termed "interrupted sensory epithelium." The distribution of VNO sensory epithelium does not appear to reflect phylogenetic influences; it is more likely a functional characteristic that varies throughout postnatal life. Therefore, this chemosensory system has a high degree of plasticity relating to age and function, which in some instances can confound the use of characteristics as phylogenetic traits. Further study is needed to quantify VNO receptors in various species to determine if functional differences exist and if some species have more precocious VNO function than others.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.     

Species-specific chemosignals evoke delayed excitation of the vomeronasal amygdala in freely-moving female rats

Male rat chemosignals attract females and influence their reproductive status. Through the accessory olfactory bulb and its projection target, the posteromedial cortical nucleus of the amygdala (PMCo), species-specific chemosignals detected by the vomeronasal organ (VNO) may reach the hypothalamus. To test this hypothesis in vivo, behavioural activation and neurotransmitter release in the PMCo were simultaneously monitored in freely moving female oestrus rats exposed to either rat or mouse urinary stimuli, or to odorants. Plasma levels of the luteinizing hormone were subsequently monitored. All stimuli induced an immediate behavioural activation, but only species-specific chemosignals led to a delayed behavioural activation. This biphasic behavioural activation was accompanied by a VNO-mediated release of the excitatory amino acids, aspartate and glutamate, in the PMCo. The late behavioural and neurochemical activation was followed by an increase in the levels of circulating luteinizing hormone. In conclusion, these data show that only species-specific chemosignals induce a delayed behavioural activation and excitatory activation of the PMCo, which is dependent on an intact VNO.     

Nursing behavior in lactating rats--the role of the vomeronasal organ

The vomeronasal organ (VNO) of female Wistar-Imamichi rats was removed completely. The females were impregnated and gave birth to young. The question addressed was whether such dams would show deficits in nursing behavior. The data indicated that removal of the VNO severely depressed nursing behavior. We conclude that the VNO contributes to the nursing behavior of lactating rats.     

Molecular biology of pheromone perception in mammals

In mammals, olfactory sensory perception is mediated by two anatomically and functionally distinct sensory organs: the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Pheromones activate the VNO and elicit a characteristic array of innate reproductive and social behaviors, along with dramatic neuroendocrine responses. Recent approaches have provided new insights into the molecular biology of sensory transduction in the vomeronasal organ. Differential screening of cDNA libraries constructed from single sensory neurons from the rat VNO has led to the isolation of a family of genes which are likely to encode mammalian pheromone receptors. The isolation of these receptors from the vomeronasal organ might permit the analysis of the molecular events which translate the bindings of pheromones into innate stereotypic behaviors and help to elucidate the logic of pheromone perception in mammals.     

Identification of non-functional human VNO receptor genes provides evidence for vestigiality of the human VNO.

In mammals, the vomeronasal organ (VNO) contains chemosensory receptor cells that bind to pheromones and induce a variety of social and reproductive behaviors. It has been traditionally assumed that the human VNO (Jacobson's organ) is a vestigial structure, although recent studies have shown minor evidence for a structurally intact and possibly functional VNO. The presence and function of the human VNO remains controversial, however, as pheromones and VNO receptors have not been well characterized. In this study we screened a human Bacterial Artificial Chromosome (BAC) library with multiple primer sets designed from human cDNA sequences homologous to mouse VNO receptor genes. Utilizing these BAC sequences in addition to mouse VNO receptor sequences, we screened the High Throughput Genome Sequence (HTGS) database to find additional human putative VNO receptor genes. We report the identification of 56 BACs carrying 34 distinct putative VNO receptor gene sequences, all of which appear to be pseudogenes. Sequence analysis indicates substantial homology to mouse V1R and V2R VNO receptor families. Furthermore, chromosomal localization via FISH analysis and RH mapping reveal that the majority of the BACs are localized to telomeric and centromeric chromosomal localizations and may have arisen through duplication events. These data yield insight into the present state of pheromonal olfaction in humans and into the evolutionary history of human VNO receptors.     

棕色田鼠雄性幼体不同发育期犁鼻器和副嗅球的组织结构

通过对出生后不同发育时期雄性棕色田鼠犁鼻器和副嗅球进行组织学观察, 探讨棕色田鼠出生后犁鼻器和副嗅球的发育规律。实验以出生后当天(0 日龄) , 5 日龄, 15 日龄, 25 日龄以及成年棕色田鼠为研究对象,副嗅球采用Pischinger 氏染色法染色, 犁鼻器用H. E. 染色法染色后进行组织学观察。结果显示, 棕色田鼠出生时, 犁鼻器和副嗅球就已具有成体的基本结构, 随着动物个体的发育, 犁鼻上皮逐渐增厚, 犁鼻管变长, 犁鼻上皮中神经元密度增加; 腺体逐渐增大, 犁鼻管腔填充物增多, 犁鼻管背外侧的静脉血管逐日增大, 管腔周围出现越来越多的血管; 副嗅球长宽都增加, 僧帽细胞层和颗粒细胞层逐渐增长, 各层细胞密度变化稍有不同;出生后15 日内, 僧帽细胞层细胞密度增加, 15 日龄以后又开始降低, 25 日龄及成体的僧帽细胞层细胞密度与5日龄的相似; 颗粒细胞层细胞密度持续增高。实验结果提示, 棕色田鼠5 日龄时, 犁鼻器和副嗅球已具有了完整的结构, 到25 日龄时可能达到了功能上的成熟。     

The vomeronasal organ and adjacent glands express components of signaling cascades found in sensory neurons in the main olfactory system

The vomeronasal organ (VNO) is a sensory organ that influences social and/or reproductive behavior and, in many cases, the survival of an organism. The VNO is believed to mediate responses to pheromones; however, many mechanisms of signal transduction in the VNO remain elusive. Here, we examined the expression of proteins involved in signal transduction that are found in the main olfactory system in the VNO. The localization of many signaling molecules in the VNO is quite different from those in the main olfactory system, suggesting differences in signal transduction mechanisms between these two chemosensory organs. Various signaling molecules are expressed in distinct areas of VNO sensory epithelium. Interestingly, we found the expressions of groups of these signaling molecules in glandular tissues adjacent to VNO, supporting the physiological significance of these glandular tissues. Our finding of high expression of signaling proteins in glandular tissues suggests that neurohumoral factors influence glandular tissues to modulate signaling cascades that in turn alter the responses of the VNO to hormonal status.     

High-resolution magnetic resonance spectroscopy of the mouse vomeronasal organ

The chemical composition of the vomeronasal organ (VNO) was investigated by means of in vitro proton magnetic resonance spectroscopy (MRS) in prepubertal and adult mice of both sexes. Results demonstrate that MRS detects several chemical constituents in the VNO, showing their age- and sex-associated changes in concentration. Preliminary experiments also suggest the ability of MRS to show compositional changes in the VNO after pheromonal stimulation. MRS can serve as a useful technique to investigate vomeronasal chemoreception.     

On the topographic targeting of basal vomeronasal axons through Slit-mediated chemorepulsion

The vomeronasal projection conveys information provided by pheromones and detected by neurones in the vomeronasal organ (VNO) to the accessory olfactory bulb (AOB) and thence to other regions of the brain such as the amygdala. The VNO-AOB projection is topographically organised such that axons from apical and basal parts of the VNO terminate in the anterior and posterior AOB respectively. We provide evidence that the Slit family of axon guidance molecules and their Robo receptors contribute to the topographic targeting of basal vomeronasal axons. Robo receptor expression is confined largely to basal VNO axons, while Slits are differentially expressed in the AOB with a higher concentration in the anterior part, which basal axons do not invade. Immunohistochemistry using a Robo-specific antibody reveals a zone-specific targeting of VNO axons in the AOB well before cell bodies of these neurones in the VNO acquire their final zonal position. In vitro assays show that Slit1-Slit3 chemorepel VNO axons, suggesting that basal axons are guided to the posterior AOB due to chemorepulsive activity of Slits in the anterior AOB. These data in combination with recently obtained other data suggest a model for the topographic targeting in the vomeronasal projection where ephrin-As and neuropilins guide apical VNO axons, while Robo/Slit interactions are important components in the targeting of basal VNO axons.     

Fine structure of the vomeronasal organ in the grass lizard, Takydromus tachydromoides

The squamates are composed of many taxa, among which there is morphological variation in the vomeronasal organ (VNO). To elucidate the evolution of chemoreception in squamate reptiles, morphological data from the VNO from a variety of squamate species is required. In this study, the morphology of the VNO of the grass lizard Takydromus tachydromoides was examined using light and electron microscopy. The VNO consists of a pair of dome-shaped structures, which communicate with the oral cavity. There are no associated glandular structures. Microvilli are present on the apical surfaces of receptor cells in its sensory epithelium, as well as on supporting cells, and there are centrioles and ciliary precursor bodies on the dendrites. In addition to ciliated cells and basal cells in the non-sensory epithelium, there is a novel type of non-ciliated cell in T. tachydromoides. They have constricted apical cytoplasm and microvilli instead of cilia, and are sparsely distributed in the epithelium. Based on these results, the variation in the morphology of the VNO in scincomorpha, a representative squamate taxon, is discussed.     

The vomeronasal organ of the male ferret.

The vomeronasal organ (VNO) is known to play a major role in sexual behavior in many mammals. This study is the first report that the adult male ferret has a VNO, which is considerably smaller and morphologically different from the usually crescent-shaped epithelium in several mammalian species, particularly rodents. There were no differences in the size or structure of the ferret VNO between the mating season in spring and the sexually quiescent season in autumn, although plasma testosterone, testis size and brain size are dramatically increased in spring and behavior changes significantly. The histological data suggest that the VNO might be not as important a structure in male ferret sexual behavior as in rodents.     

Electrophysiological and biochemical responses of mouse vomeronasal receptor cells to urine-derived compounds: possible mechanism of action

Receptor cells of the vomeronasal organ (VNO) are thought to detect pheromone-like molecules important for reproductive physiology. Several compounds derived from male mouse urine have been demonstrated to affect endocrine events in female mice. In the present study, the ability of these compounds to affect VNO activity was tested. In dissociated VNO cells held under voltage clamp conditions, application of dehydro-exo-brevicomin (DHB) evoked an outward current at negative holding potentials and an inward current at positive holding potentials. Under current clamp, DHB reduced action potential firing. Since DHB application caused a decrease in membrane conductance, this compound appeared to act by reducing inward current through closing an ion channel. Biochemical experiments tested the effects of DHB and 2- (sec-butyl)-4,5-dihydrothiazole (SBT) on cAMP levels in the VNO. A mixture of DHB and SBT decreased cAMP levels in VNO sensory tissue and had no effect on VNO non-sensory tissue. The results suggest that pheromones have an inhibitory influence on action potential generation and on cAMP levels in receptor cells of the VNO.      

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.