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.     

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.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system.

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex alpha-galactosyl and alpha-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell-cell interactions during development and maintenance of vomeronasal connections.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 1: The CC1 immunoreactive glycolipid defines a rostrocaudal gradient in the rat vomeronasal system

Primary sensory neurons in the vomeronasal organ (VNO) project axons to the glomeruli of the accessory olfactory bulb (AOB) where they form connections with mitral cell dendrites. We demonstrate here that monoclonal antibodies to specific carbohydrate antigens define stage- and position-specific events during the development of the vomeronasal system (VN). CC1 monoclonal antibodies react with specific N-acetyl galactosamine containing glycolipids. In the embryo, CC1 antigens are expressed throughout the VNO and on vomeronasal nerves. Beginning approximately at birth and continuing into adults, CC1 expression is spatially restricted in the VNO to centrally located cell bodies. In the postnatal AOB, CC1 is expressed in the nerve layer and glomeruli, but only in the rostral half of the AOB. These data suggest that CC1 antigens may participate in the targeting of axons from centrally located VNO neurons to rostral glomeruli in the AOB. In contrast, CC2 monoclonal antibodies, which recognize complex α-galactosyl and α-fucosyl glycoproteins and glycolipids, react with all VNO cell bodies and VN nerves from embryonic (E) day 15 to adults. CC2 antibodies do not distinguish rostral from caudal regions of the AOB, nor are the CC2 glycoconjugates developmentally regulated. P-Path monoclonal antibodies, which recognize 9-O-acetyl sialic acid, react with cell bodies in the VNO and nerve fibers from E13 to postnatal (P) day 2. P-Path immunoreactivity disappears from the VNO system almost completely by P14, when only a few P-Path reactive nerve fibers can be seen. These studies suggest that specific cell surface glycoconjugates may participate in spatially and temporally selective cell–cell interactions during development and maintenance of vomeronasal connections.     

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.     

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.     

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.     

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。     

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.     

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.     

Timing of neuronal intermediate filament proteins expression in the mouse vomeronasal organ during pre- and postnatal development. An immunohistochemical study

Several types of intermediate filament proteins are expressed in developing and mature neurons; they cooperate with other cytoskeletal components to sustain neuronal function from early neurogenesis onward. In this work the timing of expression of nestin, peripherin, internexin, and the neuronal intermediate filament triplet [polypeptide subunits of low (NF-L), medium (NF-M), and high (NF-H) molecular weight] was investigated in the developing fetal and postnatal mouse vomeronasal organ (VNO) by means of immunohistochemistry. The results show that the sequence of expression of intermediate filament proteins is internexin, nestin, and NF-M in the developing vomeronasal sensory epithelium; internexin, peripherin, and NF-M in the developing vomeronasal nerve; and nestin, internexin and peripherin, NF-L, and NF-M in the nerve supply to accessory structures of the VNO. At sexual maturity (2 months) NF-M is only expressed in vomeronasal neurons and NF-M, NF-L and peripherin are expressed in extrinsic nerves supplying VNO structures. The differential distribution of intermediate filament proteins in the vomeronasal sensory epithelium and nerve is discussed in terms of the cell types present therein. It is concluded that several intermediate filament proteins are sequentially expressed during intrauterine development of the VNO neural structures in a different pattern according to the different components of the VNO.     

Heterogeneity in the accessory olfactory system

The mammalian accessory olfactory bulb (AOB) is chemoarchitecturally heterogeneous in that it stains differentially with a number of markers; the receptor cells that project to the AOB are similarly heterogeneous. What is the significance of this heterogeneity? We have found that the AOB of the gray, short-tailed opossum, Monodelphis domestica, stains differentially with a number of 'markers': antibodies to olfactory marker protein (OMP) and the alpha subunit of the G protein Gi2, the lectin of Vicia villosa and NADPH-diaphorase. These markers stain the rostral AOB more strongly than the caudal AOB whereas, the G protein subunit G(o) alpha is located predominantly in the posterior subdivision of the AOB. This heterogeneity in the chemoarchitecture of the AOB may reflect a fundamental organizational dichotomy within the vomeronasal system that corresponds to a functional dichotomy. The vomeronasal sensory epithelium also exhibits a chemoarchitectural heterogeneity: receptor cells in the basal third are G(o) alpha-immunoreactive whereas the cells in the middle third are Gi2 alpha-immunoreactive. Tracing studies using WGA-HRP demonstrate that the neurons in the middle third of the vomeronasal sensory epithelium project their axons to the anterior AOB whereas those in the basal third appear to project to the posterior AOB.      

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。     

Modification of synapse formation of accessory olfactory bulb neurons by coculture with vomeronasal neurons

Previously, a coculture system of accessory olfactory bulb (AOB) neurons and vomeronasal (VN) neurons was established for studying the functional roles of AOB neurons in pheromonal signal processing. In this study, the effect of VN neurons on the development of AOB neurons was examined in a coculture system. Spine density was quantitatively measured for various culture periods of 21, 28, 36, and 42 days in vitro. The densities of dendritic spines were lower in the coculture than in single culture for all periods in vitro. Synapse formation on spines was analyzed immunocytochemically using an anti-synaptophysin antibody. The ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. The volume of spine head was larger in the coculture than in single culture. These changes were not observed in the coculture in which there was no physical contact between AOB neurons and VN neurons. These observations suggest that synapse formation on the spines of AOB neurons is modified by physical contact with VN neurons.     

A multireceptor genetic approach uncovers an ordered integration of VNO sensory inputs in the accessory olfactory bulb

Pheromone detection by the vomeronasal organ (VNO) is thought to rely on activation of specific receptors from the V1R and V2R gene families, but the central representation of pheromone receptor activation remains poorly understood. We generated transgenic mouse lines in which projections from multiple populations of VNO neurons, each expressing a distinct V1R, are differentially labeled with fluorescent proteins. This approach revealed that inputs from neurons expressing closely related V1Rs intermingle within shared, spatially conserved domains of the accessory olfactory bulb (AOB). Mitral cell-glomerular connectivity was examined by injecting intracellular dyes into AOB mitral cells and monitoring dendritic contacts with genetically labeled glomeruli. We show that individual mitral cells extend dendrites to glomeruli associated with different, but likely closely related, V1Rs. This organization differs from the labeled line of OR signaling in the main olfactory system and suggests that integration of information may already occur at the level of the AOB.     

Trpc2 gene impacts on maternal aggression, accessory olfactory bulb anatomy and brain activity

The Trpc2 gene codes for an ion channel found in the vomeronasal organ (VNO). Studies using the Trpc 2^−/− (KO) mouse have exploited the gene's role in signal transduction to explore the VNO's role in pheromonally mediated behaviors. To date, no study has evaluated the impact of the Trpc2 gene on activity within the brain. In this study, we examine the gene's effect on brain regions governing maternal aggression. We intruder-tested lactating dams and then quantified Fos immunoreactivity (Fos-IR) in the vomeronasal amygdala, hypothalamus, olfactory regions and accessory olfactory bulb (AOB). Our data confirm previous reports that loss of the Trpc2 gene severely diminishes maternal aggression. We also show that deletion of the gene results in differential hypotrophy of the glomerular layer (GlA) of the AOB, with the anterior portion the GlA resembling that of wild-type mice, and the posterior portion reduced or absent. This anatomy is suggestive of residual functioning in the apical VNO of these animals. Our Fos study describes an impact of the deletion on a network of 21 brain regions involved in emotion, aggression and olfaction, suggesting that signals from the VNO mediate activity throughout the brain. Home-cage observations of KO dams show specific deficits in nest-building, suggesting a role for pup pheromones in inducing and maintaining pup-directed maternal behaviors as well as maternal aggression.     

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.     

Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system

To date, over 100 vomeronasal receptor type 1 (V1R) genes have been identified in rodents. V1R is specifically expressed in the rodent vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. Recently, 21 putatively functional V1R genes were identified in the genome database of the amphibian Xenopus tropicalis. Amphibians are the first vertebrates to possess a VNO. In order to determine at which point during evolution the vertebrate V1R genes began to function in the vomeronasal system, we analyzed the expression of all putatively functional V1R genes in Xenopus olfactory organs. We found that V1R expression was not detected in the VNO but was specifically detected in the main olfactory epithelium (MOE). We also observed that V1R-expressing cells in the MOE coexpressed Gi2, thus suggesting that the V1R-Gi2-mediated signal transduction pathway, which is considered to play an important role in pheromone reception in the rodent VNO, exists in the amphibian MOE. These results suggest that V1R-mediated signal transduction pathway functions in Xenopus main olfactory system.     

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.     

Combinatorial coexpression of neural and immune multigene families in mouse vomeronasal sensory neurons

The vomeronasal organ (VNO) is a chemosensory organ specialized in the detection of pheromones in higher vertebrates. In mouse and rat, two gene superfamilies, V1r and V2r vomeronasal receptor genes, are expressed in sensory neurons whose cell bodies are located in, respectively, the apical and basal layers of the VNO epithelium. Here, we report that neurons of the basal layer express another multigene family, termed H2-Mv, representing nonclassical class I genes of the major histocompatibility complex. The nine H2-Mv genes are expressed differentially in subsets of neurons. More than one H2-Mv gene can be expressed in an individual neuron. In situ hybridization with probes for H2-Mv and V2r genes reveals complex and nonrandom combinations of coexpression. While neural expression of Mhc class I molecules is increasingly being appreciated, the H2-Mv family is distinguished by variegated expression across seemingly similar neurons and coexpression with a distinct multigene family encoding neural receptors. Our findings suggest that basal vomeronasal sensory neurons may consist of multiple lineages or compartments, defined by particular combinations of V2r and H2-Mv gene expression.