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.     

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.     

MHC homologs in the nervous system--they haven't lost their groove

Major histocompatibility complex (MHC) molecules have been implicated in a number of non-immune roles in the central nervous system, particularly in synaptic development and plasticity. The discovery of M10 (50% sequence identity to classical MHC molecules) proteins expressed in the vomeronasal organ adds to the list of non-traditional roles of MHC homologs. M10 molecules associate with the V2R class of vomeronasal receptors, a family of G-protein coupled receptors thought to function as pheromone receptors. Recent studies showing that classical MHC-binding peptides activate V2R-expressing neurons offer tempting clues that M10s might participate directly in the recognition of pheromone ligands, but M10 proteins do not bind to these peptides with significant affinity. Instead of presenting MHC-binding peptides, M10s might function as molecular chaperones to V2R receptors or more generally as modulators of neuronal function, as demonstrated elsewhere in the brain for classical MHC molecules.     

Structure of a pheromone receptor-associated MHC molecule with an open and empty groove

Neurons in the murine vomeronasal organ (VNO) express a family of class Ib major histocompatibility complex (MHC) proteins (M10s) that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide-binding MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I-binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8-10-mer class I-binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC-binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.     

The vomeronasal receptor V2R2 does not require escort molecules for expression in heterologous systems

In rodents, many behavioural responses are triggered by pheromones. These molecules are believed to bind and activate two families of G-protein coupled receptors, namely V1Rs and V2Rs, which are specifically expressed in the chemosensory neurons of the vomeronasal organ. V2Rs are homologous with Group 3 of G-protein-coupled receptors, which includes metabotropic glutamate receptors, calcium-sensing receptors, fish olfactory receptors, and taste receptors for sweet molecules and amino acids. The large extracellular region of these receptors is folded as a dimer and, in this form, binds agonists that in many cases are amino acids. It has recently been reported that V2Rs must be physically associated with specific major histocompatibility complex class Ib molecules (MHC) for their expression in both mouse vomeronasal neurons and heterologous cell lines. Here, we show that in contrast to the other V2Rs, V2R2, an atypical member of this receptor family, can be successfully and abundantly expressed by insect cells without the requirement of escort molecules like MHC. Moreover, the extracellular binding domain of V2R2, secreted as a soluble product, forms dimers via cysteine-mediated sulphur bridges. Overall, the data presented in this paper confirm that V2R2 diverges from the other members of the V2R family and suggest a different role for this receptor in pheromonal communication.     

A recent class of chemosensory neurons developed in mouse and rat

In most animal species, the vomeronasal organ ensures the individual recognition of conspecifics, a prerequisite for a successful reproduction. The vomeronasal organ expresses several receptors for pheromone detection. Mouse vomeronasal type-2 receptors (V2Rs) are restricted to the basal neurons of this organ and organized in four families. Family-A, B and D (family ABD) V2Rs are expressed monogenically (one receptor per neuron) and coexpress with either Vmn2r1 or Vmn2r2, two members of family-C V2Rs. Thus, basal neurons are characterized by specific combinations of two V2Rs. To investigate this issue, we raised antibodies against all family-C V2Rs and analyzed their expression pattern. We found that six out of seven family-C V2Rs (Vmn2r2-7) largely coexpressed and that none of the anti-Vmn2r2-7 antibodies significantly stained Vmn2r1 positive neurons. Thus, basal neurons are divided into two complementary subsets. The first subset (Vmn2r1-positive) preferentially coexpresses a distinct group of family-ABD V2Rs, whereas the second subset (Vmn2r2-7-positive) coexpresses the remaining group of V2Rs. Phylogenetic reconstruction and the analysis of genetic loci in various species reveal that receptors expressed by this second neuronal subset are recent branches of the V2R tree exclusively present in mouse and rat. Conversely, V2Rs expressed in Vmn2r1 positive neurons, are phylogenetically ancient and found in most vertebrates including rodents. Noticeably, the more recent neuronal subset expresses a type of Major Histocompatibility Complex genes only found in murine species. These results indicate that the expansion of the V2R repertoire in a murine ancestor occurred with the establishment of a new population of vomeronasal neurons in which coexists the polygenic expression of a recent group of family-C V2Rs (Vmn2r2-7) and the monogenic expression of a recent group of family-ABD V2Rs. This evolutionary innovation could provide a molecular rationale for the exquisite ability in individual recognition and mate choice of murine species.     

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species ^1,2. These intraspecies signals, the pheromones, as well as signals from some predators ³, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity ⁴. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR ^5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively ^6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli ^4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs ^3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations ^12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.Sensory stimulation elicits strong Ca²⁺ influx in VSNs that is indicative of receptor activation ^4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs ^15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca²⁺ imaging experiments. This method has eliminated the dye loading process used in previous studies ^4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.     

Rapid turnover and species-specificity of vomeronasal pheromone receptor genes in mice and rats

Pheromones are used by individuals of the same species to elicit behavioral or physiological changes, and they are perceived primarily by the vomeronasal organ (VNO) in terrestrial vertebrates. VNO pheromone receptors are encoded by the V1r and V2r gene superfamilies in mammals. A comparison of the V1r and V2r repertoires between closely related species can provide significant insights into the evolutionary genetic mechanisms responsible for species-specific pheromone communications. A total of 137 putatively functional V1r genes of 12 families were previously identified from the mouse genome. We report the identification of 95 putatively functional V1r genes from the draft rat genome sequence. These genes map primarily to four blocks in two chromosomes. The rat V1r genes can be phylogenetically grouped into 10 families, which are shared with mouse, and 2 new families, which are rat-specific. Even in many shared families, gene numbers differ between the two species, apparently due to frequent gene duplication and pseudogenization after the separation of the two species. Molecular dating suggests that most of the rat V1r families emerged before or during the radiation of mammalian orders, but many duplications within families occurred as recently as in the past 10 million years (MY). Our results show that the evolution of the V1r repertoire is characterized by exceptionally fast gene turnover via gains and losses of individual genes, suggesting rapid and substantial changes in pheromone communication between species.     

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.     

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.     

Composition and evolution of the V2r vomeronasal receptor gene repertoire in mice and rats

Pheromones are chemicals produced and detected by conspecifics to elicit social/sexual physiological and behavioral responses, and they are perceived primarily by the vomeronasal organ (VNO) in terrestrial vertebrates. Two large superfamilies of G protein-coupled receptors, V1rs and V2rs, have been identified as pheromone receptors in vomeronasal sensory neurons. Based on a computational analysis of the mouse and rat genome sequences, we report the first global draft of the V2r gene repertoire, composed of approximately 200 genes and pseudogenes. Rodent V2rs are subject to rapid gene births/deaths and accelerated amino acid substitutions, likely reflecting the species-specific nature of pheromones. Vertebrate V2rs appear to have originated twice prior to the emergence of the VNO in ancestral tetrapods, explaining seemingly inconsistent observations among different V2rs. The identification of the entire V2r repertoire opens the door to genomic-level studies of the structure, function, and evolution of this diverse group of sensory receptors.     

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.      

Vomeronasal organ: pheromone recognition with a twist

Pheromones are detected by the vomeronasal organ using members of two receptor superfamilies: the V1Rs and V2Rs. New studies show that MHC class I molecules are co-expressed in particular combinations with specific V2Rs in the vomeronasal organ. The role of these MHC molecules is unknown, but they may be of considerable biological significance.     

Evolutionary Patterns and Selective Pressures of Odorant/Pheromone Receptor Gene Families in Teleost Fishes

Background

Teleost fishes do not have a vomeronasal organ (VNO), and their vomeronasal receptors (V1Rs, V2Rs) are expressed in the main olfactory epithelium (MOE), as are odorant receptors (ORs) and trace amine-associated receptors (TAARs). In this study, to obtain insights into the functional distinction among the four chemosensory receptor families in teleost fishes, their evolutionary patterns were examined in zebrafish, medaka, stickleback, fugu, and spotted green pufferfish.

Methodology/Principal Findings

Phylogenetic analysis revealed that many lineage-specific gene gains and losses occurred in the teleost fish TAARs, whereas only a few gene gains and losses have taken place in the teleost fish vomeronasal receptors. In addition, synonymous and nonsynonymous nucleotide substitution rate ratios (K_A/K_S) in TAARs tended to be higher than those in ORs and V2Rs.

Conclusions/Significance

Frequent gene gains/losses and high K_A/K_S in teleost TAARs suggest that receptors in this family are used for detecting some species-specific chemicals such as pheromones. Conversely, conserved repertoires of V1R and V2R families in teleost fishes may imply that receptors in these families perceive common odorants for teleosts, such as amino acids. Teleost ORs showed intermediate evolutionary pattern between TAARs and vomeronasal receptors. Many teleost ORs seem to be used for common odorants, but some ORs may have evolved to recognize lineage-specific odors.     

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.     

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.