Encoding Olfactory Signals via Multiple Chemosensory Systems

ABSTRACT

Most animals have evolved multiple olfactory systems to detect general odors as well as social cues. The sophistication and interaction of these systems permit precise detection of food, danger, and mates, all crucial elements for survival. In most mammals, the nose contains two well described chemosensory apparatuses (the main olfactory epithelium and the vomeronasal organ), each of which comprises several subtypes of sensory neurons expressing distinct receptors and signal transduction machineries. In many species (e.g., rodents), the nasal cavity also includes two spatially segregated clusters of neurons forming the septal organ of Masera and the Grueneberg ganglion. Results of recent studies suggest that these chemosensory systems perceive diverse but overlapping olfactory cues and that some neurons may even detect the pressure changes carried by the airflow. This review provides an update on how chemosensory neurons transduce chemical (and possibly mechanical) stimuli into electrical signals, and what information each system brings into the brain. Future investigation will focus on the specific ligands that each system detects with a behavioral context and the processing networks that each system involves in the brain. Such studies will lead to a better understanding of how the multiple olfactory systems, acting in concert, offer a complete representation of the chemical world.     

Molecular and evolutionary analyses of formyl peptide receptors suggest the absence of VNO-specific FPRs in primates

Formyl peptide receptors (FPRs) were observed to expand in rodents and were recently suggested as candidate vomeronasal chemo-sensory receptors. Since vomeronasal chemosensory receptors usually underwent positive selection and evolved concordantiy with the vomeronasal organ (VNO) morphology, we surveyed FPRs in primates in which VNO morphology is greatly diverse and thus it would provide us a clearer view of VNO-FPRs evolution. By screening available primate genome sequences, we obtained the FPR repertoires in representative primate species. As a result, we did not find FPR family size expansion in primates. Further analyses showed no evolutionary force variance between primates with or without VNO structure, which indicated that there was no functional divergence among primates FPRs. Our results suggest that primates lack the VNO-specific FPRs and the FPR expansion is not a common phenomenon in mammals outside rodent lineage, regardless of VNO complexity.     

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.     

Olfactory receptors: G protein-coupled receptors and beyond

Sensing the chemical environment is critical for all organisms. Diverse animals from insects to mammals utilize highly organized olfactory system to detect, encode, and process chemostimuli that may carry important information critical for health, survival, social interactions and reproduction. Therefore, for animals to properly interpret and react to their environment it is imperative that the olfactory system recognizes chemical stimuli with appropriate selectivity and sensitivity. Because olfactory receptor proteins play such an essential role in the specific recognition of diverse stimuli, understanding how they interact with and transduce their cognate ligands is a high priority. In the nearly two decades since the discovery that the mammalian odorant receptor gene family constitutes the largest group of G protein-coupled receptor (GPCR) genes, much attention has been focused on the roles of GPCRs in vertebrate and invertebrate olfaction. However, is has become clear that the 'family' of olfactory receptors is highly diverse, with roles for enzymes and ligand-gated ion channels as well as GPCRs in the primary detection of olfactory stimuli.     

Expression of cGMP signaling elements in the Grueneberg ganglion

The Grueneberg ganglion (GG) is a cluster of neurons localized to the vestibule of the anterior nasal cavity. Based on axonal projections to the olfactory bulb of the brain, as well as expression of olfactory receptors and the olfactory marker protein, it is considered a chemosensory subsystem. Recently, it was observed that in mice, GG neurons respond to cool ambient temperatures. In mammals, coolness-induced responses in highly specialized neuronal cells are supposed to rely on the ion channel TRPM8, whereas in thermosensory neurons of the nematode worm Caenorhabditis elegans, detection of environmental temperature is mainly mediated by cyclic guanosine monophosphate (cGMP) pathways, in which cGMP is generated by transmembrane guanylyl cyclases. To unravel the molecular mechanisms underlying coolness-induced responses in GG neurons, potential expression of TRPM8 in the murine GG was investigated; however, no evidence was found that this ion channel is present in the GG. By contrast, a substantial number of GG neurons was observed to express the transmembrane guanylyl cyclase subtype GC-G. In the nose, GC-G expression appears to be confined to the GG since it was not detectable in other nasal compartments. In the GG, coolness-stimulated responses are only observed in neurons characterized by the expression of the olfactory receptor V2r83. Interestingly, expression of GC-G in the GG was found in this V2r83-positive subpopulation but not in other GG neurons. In addition to GC-G, V2r83-positive GG cells also co-express the phosphodiesterase PDE2A. Thus, in summary, coolness-sensitive V2r83-expressing GG neurons are endowed with a cGMP cascade which might underlie thermosensitivity of these cells, similar to the cGMP pathway mediating thermosensation in neurons of C. elegans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Fleischer and K. Mamasuew contributed equally to this work.     

离体培养嗅鞘细胞促进新生大鼠螺旋神经节细胞的存活

本文旨在探索离体实验中的嗅鞘细胞(olfactory ensheathing cells,OECs)有无促进耳蜗听觉传入神经元——螺旋神经节细胞(spiral ganglion cells,SGCs)存活作用及其可能机制。取成年大鼠嗅球和新生大鼠蜗轴组织块进行OECs与SGCs的培养,采用差速贴壁法纯化培养OECs。实验分OECs与SGCs共培养组和SGCs单独培养组。倒置相差显微镜下观察OECs和SGCs生长状态,神经营养因子受体p75免疫组织化学法鉴定OECs,神经元特异性标志物βIII-tubulin标记SGCs。为了研究OECs与SGCs共培养体系中,前者促进后者存活的可能机制,共培养组中分别加入脑源性神经生长因子(BDNF,500pg/mL)和BDNF抗体(IgY型,50μg/mL),对照组为未加任何处理的共培养组,然后检查各培养组中SGCs存活数量和存活时间。结果显示,OECs贴壁培养7d后形成一细胞单层,在OECs与SGCs共培养体系中,SGCs在OECs形成的细胞单层的表面生长,并伸出长突起,呈现典型的双极神经元形态;在培养的前6天内,随着培养时间的增加,两组中的SGCs都较接种前减少,但共培养组中SGCs存活数量明显高于SGCs单独培养组(P0.01);单独培养组的SGCs数量在培养的第6天出现大幅度减少,在培养的第9天几乎没有生长;共培养组的SGCs数量未见明显变化(P0.05);共培养中加入BDNF对OECs促进SGCs存活无明显影响,而加入BDNF抗体(IgY)后存活的SGCs减少(P0.01)。本研究结果提示,OECs与SGCs共培养能够促进新生大鼠SGCs存活和突起生长,延长存活时间,OECs分泌BDNF可能是促进SGCs存活的机制之一。     

Histological Architecture of the Nasal Region in Rana chensinensis

The nasal region of Rana chensinensis is divided into the nasal capsules and nasal cavities. In this study, we investigated the adult histological structure of the nasal capsules and nasal cavities in the frog R. chensinensis under the microscope. We found that an eminentia olfactoria is present in this frog and the presence of the eminentia olfactoria may be connected to a terrestrial life style. The double staining method using alcian blue and alizarin red showed that the septomaxilla, the most important bone associated with the olfactory capsules, is an intramembranous bone in R. chensinensis. The opening of the nasolacrimal duct showed a close proximity to the apertura nasalis externa. The presence of the nasolacrimal duct in the olfactory region may be an adaptation to a terrestrial environment. The function of the vomeronasal and olfactory organs is discussed in the paper.     

Chiropteran vomeronasal complex and the interfamilial relationships of bats

Within the extant orders of living mammals, the distribution of the vomeronasal organ (VNO) and associated structures is very stable, being universally present in the vast majority or universally absent in cetaceans and sirenians. Chiroptera is the most noteworthy exception, with variation in the absence or presence of the vomeronasal complex occurring even at the species level in some instances. The VNO and/or its component structures, such as the accessory olfactory bulb, were studied in serially sectioned snouts and brains from 114 genera and 292 species representing all extant chiropteran families except Myzopodidae and Antrozoidae. Taxa were scored for the following characters: (1) degree of formation of the vomeronasal epithelial tube, (2) shape of the vomeronasal cartilage, (3) occurrence of the nasopalatine duct, and (4) occurrence of the accessory olfactory bulb. To reconstruct the evolutionary history of the bat vomeronasal complex, the distributions of these four characters were mapped, using the computer program MacClade, onto chiropteran phylogenies in the literature derived from other data sets. In all phylogenies, these four characters exhibit a high degree of homoplasy, only part of which is accounted for by several polymorphic taxa. However, perhaps the most remarkable result is that in the most parsimonious solutions the absence of the vomeronasal epithelial tube and accessory olfactory bulb is identified as primitive for Chiroptera, with both structures reevolving numerous times: such a scenario would be unique to bats among mammals. An alternative, though less parsimonious interpretation, which does not require reevolution of this very complex system, is that a well-developed vomeronasal epithelial tube is primitive for Chiroptera, as in nearly all other orders of mammals, but has been reduced or lost in the majority of families. Explication of the peculiar evolutionary history of the vomeronasal system in bats awaits studies on the adult morphology in the more than 630 species not yet examined and, in particular, on ontogeny, which to date is known for only a handful of taxa.A preliminary account of this research was presented at the Tenth International Bat Research Conference and Twenty-Fifth North American Bat Research Symposium held at Boston University, Massachusetts, on 6–11 August 1995.     

Firing properties of accessory olfactory bulb mitral/tufted cells in response to urine delivered to the vomeronasal organ of gray short-tailed opossums

In comparison with many mammals, there is limited knowledge of the role of pheromones in conspecific communication in the gray short-tailed opossum. Here we report that mitral/tufted (M/T) cells of the accessory olfactory bulb (AOB) of male opossums responded to female urine but not to male urine with two distinct patterns: excitation followed by inhibition or inhibition. Either pattern could be mimicked by application of guanosine 5'-O-3-thiotriphosphate and blocked by guanosine 5'-O-2-thiodiphosphate, indicating that the response of neurons in this pathway is through a G-protein-coupled receptor mechanism. In addition, the inhibitor of phospholipase C (PLC), U73122, significantly blocked urine-induced responses. Male and female urine were ineffective as stimuli for M/T cells in the AOB of female opossums. These results indicate that urine of diestrous females contains a pheromone that directly stimulates vomeronasal neurons through activation of PLC by G-protein-coupled receptor mechanisms and that the response to urine is sexually dimorphic.     

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (<1 mm³) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.     

Co-expression of mu and delta opioid receptors as receptor-G protein fusions enhances both mu and delta signalling via distinct mechanisms

Mu and delta opioid receptors (MORs and DORs) were co-expressed as fusion proteins between a receptor and a pertussis insensitive mutant Gα_i/o protein in human embryonic kidney 293 cells. Signalling efficiency was then monitored following inactivation of endogenous Gα_i/o proteins by pertussis toxin. Co-expression resulted in increased delta opioid signalling which was insensitive to the mu specific antagonist d -Phe-Cys-Tyr- d -Trp-Arg-Thr-Pen-Thr-NH₂. Under these conditions, mu opioid signalling was also increased and insensitive to the delta specific antagonist Tic-deltorphin. In this latter case, however, no G protein activation was observed in the presence of the delta specific inverse agonist N , N (CH3)₂-Dmt-Tic-NH₂. When a MOR fused to a non-functional Gα subunit was co-expressed with the DOR-Gα protein fusion, delta opioid signalling was not affected whereas mu opioid signalling was restored. Altogether our results suggest that increased delta opioid signalling is due to enhanced DOR coupling to its tethered Gα subunit. On the other hand, our data indicate that increased mu opioid signalling requires an active conformation of the DOR and also results in activation of the Gα subunit fused the DOR.     

An enhanced olfactory marker protein immunoreactivity in individual olfactory receptor neurons following olfactory bulbectomy may be related to increased neurogenesis

Olfactory marker protein (OMP) is a 19-kD acidic protein found throughout the cytoplasm of mature olfactory receptor neurons (ORNs). Its function remains unknown. Following olfactory bulbectomy, the proportion of ORNs mature enough to express OMP declines greatly. However, in the few remaining mature ORNs, it has been observed that the intensity of OMP immunoreactivity (IR) appears to increase over that of ORNs on the unoperated side. We have now investigated this phenomenon quantitatively in rats subjected to unilateral olfactory bulbectomy. Results show that at all postbulbectomy survival periods examined quantitatively (3 days to 6 months), a significant decrease (19–37%) occurs in the transmission of incident light through OMP(+)-ORNs in bulbectomized versus unoperated olfactory epithelium (OE). Further, we also observed a consistent side-to-side difference in OMP IR in control unoperated animals. Possible explanations for these observations and their relation to the still unknown function of OMP are discussed. To test the possibility that OMP might serve a mitogenic role in the OE, recombinant OMP was added to organotypic explant cultures of fetal olfactory mucosa. Addition of OMP resulted in a dose-dependent increase in the density of bromodeoxyuridine-positive cells in the cultures, with a 50% increase occurring at the plateau OMP concentration of 25 nM. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 377–390, 1998     

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 receptor and binding protein interplay in the detection of a distinct pheromone component in the silkmoth Antheraea polyphemus

Male moths respond to conspecific female-released pheromones with remarkable sensitivity and specificity, due to highly specialized chemosensory neurons in their antennae. In Antheraea silkmoths, three types of sensory neurons have been described, each responsive to one of three pheromone components. Since also three different pheromone binding proteins (PBPs) have been identified, the antenna of Antheraea seems to provide a unique model system for detailed analyzes of the interplay between the various elements underlying pheromone reception. Efforts to identify pheromone receptors of Antheraea polyphemus have led to the identification of a candidate pheromone receptor (ApolOR1). This receptor was found predominantly expressed in male antennae, specifically in neurons located beneath pheromone-sensitive sensilla trichodea. The ApolOR1-expressing cells were found to be surrounded by supporting cells co-expressing all three ApolPBPs. The response spectrum of ApolOR1 was assessed by means of calcium imaging using HEK293-cells stably expressing the receptor. It was found that at nanomolar concentrations ApolOR1-cells responded to all three pheromones when the compounds were solubilized by DMSO and also when DMSO was substituted by one of the three PBPs. However, at picomolar concentrations, cells responded only in the presence of the subtype ApolPBP2 and the pheromone (E,Z)-6,11-hexadecadienal. These results are indicative of a specific interplay of a distinct pheromone component with an appropriate binding protein and its related receptor subtype, which may be considered as basis for the remarkable sensitivity and specificity of the pheromone detection system.     

Removal of the vomeronasal organ blocks the stress-induced hyperthermia response to alarm pheromone in male rats

Previously, we reported that male Wistar rats release alarm pheromone from their perianal region, which aggravates stress-induced hyperthermia (SIH) in pheromone-recipient rats. The subsequent discovery that this pheromone could be trapped in water enabled us to expose recipients to the pheromone in their home cages. Despite its apparent influence on autonomic and behavioral functions, we still had no clear evidence as to whether the alarm pheromone was perceived by the main olfactory system (MOS) or by the vomeronasal system. In this study, we investigated this question by exposing 3 types of recipients to alarm pheromone in their home cages: intact males (Intact), vomeronasal organ-excised males (VNX), and sham-operated males (Sham). The Intact and Sham recipients showed aggravated SIH in response to alarm pheromone, whereas the VNX recipients did not. In addition, the results of the habituation/dishabituation test and soybean agglutinin binding to the accessory olfactory bulb verified the complete ablation of the vomeronasal organ (VNO) with a functional MOS in the pheromone recipients. These results strongly suggest that male rats perceive alarm pheromone with the VNO.     

Olfactory marker protein (OMP) exhibits a beta-clam fold in solution: implications for target peptide interaction and olfactory signal transduction

Olfactory marker protein (OMP) is a ubiquitous, cytoplasmic protein found in mature olfactory receptor neurons of all vertebrates. Electrophysiological and behavioral studies demonstrate that it is a modulator of the olfactory signal transduction pathway. Here, we demonstrate that the solution structure of OMP, as determined by NMR studies, is a single globular domain protein comprised of eight beta-strands forming two beta-sheets oriented orthogonally to one another, thus exhibiting a "beta-clam" or "beta-sandwich" fold: beta-sheet 1 is comprised of beta3-beta8-beta1-beta2 and beta-sheet 2 contains beta6-beta5-beta4-beta7. Insertions include two, long alpha-helices located on opposite sides of the beta-clam and three flexible loops. The juxtaposition of beta-strands beta6-beta5-beta4-beta7-beta2-beta1-beta8-beta3 forms a continuously curved surface and encloses one side of the beta-clam. The "cleft" formed by the two beta-sheets is opposite to the closed end of the beta-clam. Using a peptide titration series, we have identified this cleft as the binding surface for a peptide derived from the Bex1 protein. The highly conserved Omega-loop structure adjacent to the Bex1 peptide-binding surface found in OMP may be the site of additional OMP-protein interactions related to its role in modulating olfactory signal transduction. Thus, the interaction between the OMP and Bex1 proteins could facilitate the interaction between OMP and other components of the olfactory signaling pathway.     

Scanning mutagenesis of regions in the Gα protein Gpa1 that are predicted to interact with yeast mating pheromone receptors

The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Gα protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Gα are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (β2–β3, α2–β4, α3–β5, and α4–β6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gβγ. However, the constitutive activity caused by the F344C and E335C mutations in the α2–β4 loop and F378C in the α3–β5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gβγ. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the β2–β3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Gα contribute to activation of signaling.     

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.