Goα expression in the vomeronasal organ and olfactory bulb of the tammar wallaby

The vomeronasal organ (VNO) detects pheromones via 2 large families of receptors: vomeronasal receptor 1, associated with the protein Giα2, and vomeronasal receptor 2, associated with Goα. We investigated the distribution of Goα in the developing and adult VNO and adult olfactory bulb of a marsupial, the tammar wallaby. Some cells expressed Goα as early as day 5 postpartum, but by day 30, Goα expressing cells were distributed throughout the receptor epithelium of the VNO. In the adult tammar, Goα appeared to be expressed in sensory neurons whose nuclei were mostly basally located in the vomeronasal receptor epithelium. Goα expressing vomeronasal receptor cells led to all areas of the accessory olfactory bulb (AOB). The lack of regionally restricted projection of the vomeronasal receptor cell type 2 in the tammar was similar to the uniform type, with the crucial difference that the uniform type only shows expression of Giα2 and no expression of Goα. The observed Goα staining pattern suggests that the tammar may have a third accessory olfactory type that could be intermediate to the segregated and uniform types already described.     

Olfactory receptors and signalling elements in the Grueneberg ganglion

The Grueneberg ganglion (GG) is a cluster of neurones present in the vestibule of the anterior nasal cavity. Although its function is still elusive, recent studies have shown that cells of the GG transcribe the gene encoding the olfactory marker protein (OMP) and project their axons to glomeruli of the olfactory bulb, suggesting that they may have a chemosensory function. Chemosensory responsiveness of olfactory neurones in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO) is based on the expression of either odorant receptors or vomeronasal putative pheromone receptors. To scrutinize its presumptive olfactory nature, the GG was assessed for receptor expression by extensive RT-PCR analyses, leading to the identification of a distinct vomeronasal receptor which was expressed in the majority of OMP-positive GG neurones. Along with this receptor, these cells expressed the G proteins Go and Gi, both of which are also present in sensory neurones of the vomeronasal organ. Odorant receptors were expressed by very few cells during prenatal and perinatal stages; a similar number of cells expressed adenylyl cyclase type III and G(olf/s), characteristic signalling elements of the main olfactory system. The findings of the study support the notion that the GG is in fact a subunit of the complex olfactory system, comprising cells with either a VNO-like or a MOE-like phenotype. Moreover, expression of a vomeronasal receptor indicates that the GG might serve to detect pheromones.     

Projection pattern of vomeronasal neurons to the accessory olfactory bulb in goats

Goats have a well-developed vomeronasal (VN) system and exhibit pheromone-induced reproductive facilitation, but there are no reports on the projection pattern of VN neurons in this species. Rodent, guinea pig and opossum accessory olfactory bulbs (AOBs) have been shown to have a segregated pattern of projection of the VN neurons, which express the two alpha-subtypes of the G-protein, namely Gi2 and Go, to the rostral and caudal regions of the AOB, respectively. In this study we investigated the projection pattern of VN nerve terminals by immunocytochemical staining of the goat vomeronasal organ (VNO) and the AOB with antibodies to Gi2 and Go. Gi2-immunoreactivity was found on the luminal surface of the sensory epithelium of the VNO, and in the VN nerve and glomerular layer throughout the AOB. On the other hand, Go-immunoreactivity was not identified in either the VNO or the VN nerve layer of the AOB. These results indicate that the projection pattern of VN neurons from the VNO to the AOB in the goat is considerably different from that in rodents which show a distinct segregated pattern.     

Morphological evidence for two types of Mammalian vomeronasal system

The vomeronasal (VN) systems of rodents and opossums are of the segregated type, i.e alpha-subtype G protein Gi2- or Go-expressing VN neurons, which are sensory cells, project discretely to the rostral or caudal region of the accessory olfactory bulb (AOB). Although this zone-specific projection is believed to be a common feature for processing pheromones in mammals, we previously found a uniform-type VN system in goat in which only Gi2-expressing VN axons terminate at the AOB. In most mammals, it remains unclear whether their VN systems are of the segregated or uniform type. Therefore, we investigated morphologically the VN systems of different mammalian species (dog, horse, musk shrew and common marmoset). Consequently, all VN axons of the examined animals were positively stained with immunohistochemistry for Gi2 in the same way as that in the goat. On the other hand, we observed immunoreactivities against Go in the olfactory axons, but not in the VN axons. These results suggest that many mammals have uniform-type VN systems, and at least two types of VN systems exist in terrestrial mammals. This morphological evidence will help us determine the processing function of VN systems.     

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.     

Development of olfactory marker protein and N-CAM expression in chemosensory systems of the opossum,Monodelphis domestica

Using olfactory marker protein (OMP) and neural cell adhesion molecule (N-CAM) immunohistochemistry, the present study describes development of olfactory and vomeronasal systems in pre- and postnatal opossums, Monodelphis domestica. As revealed by OMP expression, development of the main olfactory system precedes that of the vomeronasal system by 1–2 weeks. OMP is expressed throughout (homogeneously) the nerve and glomerular layers of the main (MOB) but is expressed more strongly (heterogeneously) in the anterior as compared to the posterior accessory (AOB) olfactory bulb. N-CAM expression is homogeneous in both MOB and AOB. The heterogeneity in the AOB is developmentally regulated, since in the 30-day-old AOB the expression of OMP is homogeneous, becoming heterogeneous during the second month of life. Maximal expression of N-CAM precedes maximal expression of OMP in the VNS by about 2 weeks. From 7 to 21 days of age only, there is a small population of OMP-positive, N-CAM-negative olfactory and vomeronasal axon terminals that penetrate deep into the brain parenchyma, overgrowing their normal targets in the MOB and AOB, respectively. J. Morphol. 234:109–129, 1997. © 1997 Wiley-Liss, Inc.     

A divergent pattern of sensory axonal projections is rendered convergent by second-order neurons in the accessory olfactory bulb

The mammalian vomeronasal system is specialized in pheromone detection. The neural circuitry of the accessory olfactory bulb (AOB) provides an anatomical substrate for the coding of pheromone information. Here, we describe the axonal projection pattern of vomeronasal sensory neurons to the AOB and the dendritic connectivity pattern of second-order neurons. Genetically traced sensory neurons expressing a given gene of the V2R class of vomeronasal receptors project their axons to six to ten glomeruli distributed in globally conserved areas of the AOB, a theme similar to V1R-expressing neurons. Surprisingly, second-order neurons tend to project their dendrites to glomeruli innervated by axons of sensory neurons expressing the same V1R or the same V2R gene. Convergence of receptor type information in the olfactory bulb may represent a common design in olfactory systems.     

Zonal organization of the mammalian main and accessory olfactory systems

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.     

Differential localization of G proteins, Gi and Go, in the olfactory epithelium and the main olfactory bulb of the rat.

The immunohistochemical localization of proteins Gi1 (plus Gi3). Gi2 and Go was studied in the olfactory epithelium and the main olfactory bulb of rats, using purified antibodies to the respective alpha subunits and beta gamma subunits of these G proteins. In the olfactory epithelium, only a restricted population of olfactory cells was immunopositive for Gi2 alpha, but others were not. The immunoreactivity for Gi1 alpha/Gi3 alpha was not observed. The olfactory epithelium was immunopositive for both Go alpha and beta gamma, but its apical surface was immunopositive only for beta gamma. In the main olfactory bulb, all layers were intensely immunopositive for Go alpha and beta gamma but weakly for Gi2 alpha. In contrast to the negative or weak immunostainings in the olfactory nerve fiber layer and glomeruli, the molecular and the internal granular layers were intensely immunopositive for Gi1 alpha/Gi3 alpha. These findings suggest the functional difference among Gi1/Gi3, Gi2 and Go in the signal transduction in the olfactory system.     

Deterioration of the Gαo vomeronasal pathway in sexually dimorphic mammals

In mammals, social and sexual behaviours are largely mediated by the vomeronasal system (VNS). The accessory olfactory bulb (AOB) is the first synaptic locus of the VNS and ranges from very large in Caviomorph rodents, small in carnivores and ungulates, to its complete absence in apes, elephants, most bats and aquatic species. Two pathways have been described in the VNS of mammals. In mice, vomeronasal neurons expressing Gαi2 protein project to the rostral portion of the AOB and respond mostly to small volatile molecules, whereas neurons expressing Gαo project to the caudal AOB and respond mostly to large non-volatile molecules. However, the Gαo-expressing pathway is absent in several species (horses, dogs, musk shrews, goats and marmosets) but no hypotheses have been proposed to date to explain the loss of that pathway. We noted that the species that lost the Gαo pathway belong to Laurasiatheria and Primates lineages, both clades with ubiquitous sexual dimorphisms across species. To assess whether similar events of Gαo pathway loss could have occurred convergently in dimorphic species we studied G-protein expression in the AOB of two species that independently evolved sexually dimorphic traits: the California ground squirrel Spermophilus beecheyi (Rodentia; Sciurognathi) and the cape hyrax Procavia capensis (Afrotheria; Hyracoidea). We found that both species show uniform expression of Gαi2-protein throughout AOB glomeruli, while Gαo expression is restricted to main olfactory glomeruli only. Our results suggest that the degeneration of the Gαo-expressing vomeronasal pathway has occurred independently at least four times in Eutheria, possibly related to the emergence of sexual dimorphisms and the ability of detecting the gender of conspecifics at distance.     

Selective G protein beta gamma-subunit compositions mediate phospholipase C activation in the vomeronasal organ

Chemosensory neurons of the vomeronasal organ (VNO) are supposed to detect pheromones controlling social and reproductive behavior in most terrestrial vertebrates. Recent studies indicate that pheromone signaling in VNO neurons is mediated via phospholipase C (PLC) activation generating the two second messengers inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Since G alpha(i) and G alpha(o) predominantly expressed in VNO neurons are usually not involved in activating PLC, it was explored if PLC activation may be mediated by G beta gamma subunits. It was found that a scavenger for beta gamma dimers reduced the urine-induced IP3 formation in VNO preparations in a dose-dependent manner indicating a role for G beta gamma complexes. Towards an identification of the relevant G beta and G gamma subunit(s), PCR approaches as well as immunohistochemical experiments were performed. It was found that out of the five known G beta subtypes, only G beta2 was expressed in both G alpha(i) as well as G alpha(o) neurons. Experimental approaches focusing on the spatial expression profile of identified G gamma subtypes revealed that G gamma8-positive neurons are preferentially localized to the basal region of the vomeronasal epithelium, whereas G gamma2-reactive cells are restricted to the apical G alpha(i)-positive layer of the sensory epithelium. As IP3 formation induced upon stimulation with volatile urinary compounds was selectively blocked by G gamma2-specific antibodies whereas second messenger formation elicited upon stimulation with alpha2u globulin was inhibited by antibodies recognizing G gamma8, it is conceivable that PLC activation in the two populations of chemosensory VNO neurons is mediated by different G beta gamma complexes.     

Sexual experience does not compensate for the disruptive effects of zinc sulfate--lesioning of the main olfactory epithelium on sexual behavior in male mice

Recent studies point to an important role for the main olfactory epithelium (MOE) in regulating sexual behavior in male mice. We asked whether sexual experience could compensate for the disruptive effects of lesioning the MOE on sexual behavior in male mice. Male mice, which were either sexually naive or experienced, received an intranasal irrigation of either a zinc sulfate solution to destroy the MOE or saline. Sexual behavior in mating tests with an estrous female was completely abolished in zinc sulfate-treated male mice regardless of whether subjects were sexually experienced or not before the treatment. Furthermore, zinc sulfate treatment clearly disrupted olfactory investigation of both volatile and nonvolatile odors. Destruction of the MOE by zinc sulfate treatment was confirmed by a significant reduction in the expression of Fos protein in the main olfactory bulb following exposure to estrous female urine. By contrast, vomeronasal function did not seem to be affected by zinc sulfate treatment: nasal application of estrous female urine induced similar levels of Fos protein in the mitral and granule cells of the accessory olfactory bulb (AOB) of zinc sulfate- and saline-treated males. Likewise, the expression of soybean agglutinin, which stains the axons of vomeronasal organ neurons projecting to the glomerular layer of the AOB, was similar in zinc sulfate- and saline-treated male mice. These results show that the main olfactory system is essential for the expression of sexual behavior in male mice and that sexual experience does not overcome the disruptive effects of MOE lesioning on this behavior.     

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.     

Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons

The mechanisms that underlie axonal pathfinding of vomeronasal neurons from the vomeronasal organ (VNO) in the periphery to select glomeruli in the accessory olfactory bulb (AOB) are not well understood. Neuropilin-2, a receptor for secreted semaphorins, is expressed in V1R- and V3R-expressing, but not V2R-expressing, postnatal vomeronasal neurons. Analysis of the vomeronasal nerve in neuropilin-2 (npn-2) mutant mice reveals pathfinding defects at multiple choice points. Vomeronasal sensory axons are severely defasciculated and a subset innervates the main olfactory bulb (MOB). While most axons of V1R-expressing neurons reach the AOB and converge into distinct glomeruli in stereotypic locations, they are no longer restricted to their normal anterior AOB target zone. Thus, Npn-2 and candidate pheromone receptors play distinct and complementary roles in promoting the wiring and patterning of sensory neurons in the accessory olfactory system.     

Vomeronasal epithelial cells of human fetuses contain immunoreactivity for G proteins, Go(alpha) and Gi(alpha 2).

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.     

达乌尔黄鼠犁鼻器和副嗅球的组织结构及嗅球c-Fos表达的季节变化

啮齿动物的犁鼻器和副嗅球与社会通讯和生殖行为有关,主嗅球影响其觅食行为。达乌尔黄鼠（Spermophilus dauricus）是一种具有较低社会行为的储脂类冬眠动物。本研究用组织学和免疫组织化学方法探究了其犁鼻器和副嗅球的结构特点及嗅球神经元活动对季节变化的适应。结果发现,达乌尔黄鼠犁鼻器具有较大的血管,犁鼻器管腔外侧为非感觉性的呼吸上皮（Respiratory epithelium,RE）,内侧为感觉上皮（Sensory epithelium,SE）,RE较SE薄,靠近管腔处为假复层柱状上皮。选取犁鼻器中间部位比较,发现SE的厚度、长度及感觉细胞密度均无性别差异。副嗅球位于主嗅球后方背内侧,由6层细胞构成。侧嗅束穿过副嗅球,位于颗粒细胞层之上。雄性达乌尔黄鼠较雌性有更长的僧帽细胞层和颗粒细胞层。春季（3月）和冬季（1月）达乌尔黄鼠主嗅球的嗅小球层、僧帽细胞层和颗粒细胞层的c-Fos-ir神经元密度显著低于夏季（7月）和秋季（10月）,且冬季外网织层的c-Fos-ir神经元密度显著低于夏季和秋季,说明达乌尔黄鼠在冬季和春季的嗅觉神经活动较弱,呈现出对冬眠的生理性适应。这些结果丰富了动物犁鼻器和副嗅球的形态学资料,并有助于理解冬眠动物嗅觉系统对季节变化和冬眠的适应。     

Vomeronasal phenotype and behavioral alterations in G alpha i2 mutant mice

Several social and reproductive behaviors are under the influence of the vomeronasal (VN) organ; VN neurons detect odorous molecules emitted by individuals of the same species. There are two types of VN neurons, and these differ in their expression of chemosensory receptors and G protein subunits. The significance of this dichotomy is largely unknown. VN neurons express high levels of either G alpha i2 or G alpha o. A mouse line carrying a targeted disruption of the G alpha i2 gene offered the opportunity for studying the effects of a lack of receptor signaling through the heterotrimeric Gi2 protein in one VN cell type. As a consequence of this deficiency, the number of VN neurons that normally express G alpha i2 is decreased by half. These residual neurons are defective in eliciting a response in their target neurons in the accessory olfactory bulb. Moreover, G alpha i2 mutant mice show alterations in behaviors for which an intact VN organ is known to be important. Display of maternal aggressive behavior is severely blunted, and male mice show significantly less aggression toward an intruder. However, male mice show unaltered sexual-partner preference. This suggests that the two types of VN neurons may have separate functions in mediating behavioral changes in response to chemosensory information.     

Cell turnover in the vomeronasal epithelium: evidence for differential migration and maturation of subclasses of vomeronasal neurons in the adult opossum

Previous investigations of cell turnover in the mammalian vomeronasal sensory epithelium (VN-SE) raised two issues. First, if, in addition to the already demonstrated vertical migration, horizontal migration from the edges of the VN-SE participates in neuronal replacement. Second, whether or not migration and maturation is differential in upper and lower populations of vomeronasal neurons, since these two cell populations are chemically, physiologically, functionally, and perhaps evolutionarily different. By injecting bromodeoxyuridine (BrdU) into adult opossum (Monodelphis domestica) and permitting different survival times, the pattern of distribution of BrdU-labeled cells was analyzed. No evidence of horizontal migration in neuronal replacement was found. To investigate vertical migration and maturation of subclasses of vomeronasal neurons, double immunohistochemistry of BrdU and markers of the lower (G(oalpha) protein) and upper [G(i2alpha) protein and olfactory marker protein (OMP)] cell populations were performed. Three days after administration of BrdU, some mature neurons were observed in both lower and upper layers of the VN-SE, as demonstrated by coexpression of BrdU with G(oalpha) protein and OMP, respectively. The data on vertical distribution, however, indicate that most of the daughter cells enter the G(oalpha)-protein-expressing zone of the VN-SE by day 5, whereas most daughter cells do not reach the G(i2alpha)-protein-expressing zone until day 7, suggesting that these two populations mature at slightly different rates. These results are the first evidence of differential neurogenesis of subclasses of vomeronasal neurons.     

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.