Vomeronasal Receptor Neurons Research Articles

Prosaposin/Saposin Expression in the Developing Rat Olfactory and Vomeronasal Epithelia

Prosaposin is a glycoprotein widely conserved in vertebrates, and it acts as a precursor for saposins that accelerate hydrolysis in lysosomes or acts as a neurotrophic factor without being processed into saposins. Neurogenesis in the olfactory neuroepithelia, including the olfactory epithelium (OE) and the vomeronasal epithelium (VNE), is known to occur throughout an animal’s life, and mature olfactory neurons (ORNs) and vomeronasal receptor neurons (VRNs) have recently been revealed to express prosaposin in the adult olfactory organ. In this study, the expression of prosaposin in the rat olfactory organ during postnatal development was examined. In the OE, prosaposin immunoreactivity was observed in mature ORNs labeled using olfactory marker protein (OMP) from postnatal day (P) 0. Immature ORNs showed no prosaposin immunoreactivity throughout the examined period. In the VNE, OMP-positive VRNs were mainly observed in the basal region of the VNE on P10 and showed an adult-like distribution from P20. On the other hand, prosaposin immunoreactivity was observed in VRNs from P0, suggesting that not only mature VRNs but also immature VRNs express prosaposin. This study raises the possibility that prosaposin is required for the normal development of the olfactory organ and has different roles in the OE and the VNE.

Expression patterns of prosaposin and its receptors, G protein-coupled receptor (GPR) 37 and GPR37L1, in the mouse olfactory organ

Prosaposin is a glycoprotein conserved widely in vertebrates, because it is a precursor for saposins that are required for normal lysosomal function and thus for autophagy, and acts as a neurotrophic factor. Most tetrapods possess two kinds of olfactory neuroepithelia, namely, the olfactory epithelium (OE) and the vomeronasal epithelium (VNE). This study examined the expression patterns of prosaposin and its candidate receptors, G protein-coupled receptor (GPR) 37 and GPR37L1, in mouse OE and VNE by immunofluorescence and in situ hybridization. Prosaposin immunoreactivity was observed in the olfactory receptor neurons, vomeronasal receptor neurons, Bowman’s gland (BG), and Jacobson’s gland (JG). Prosaposin expression was mainly observed in mature neurons. Prosaposin mRNA expression was observed not only in these cells but also in the apical region of the VNE. GPR37 and GPR37L1 immunoreactivities were found only in the BG and/or the JG. Prosaposin was suggested to secrete and facilitate the autophagic activities of the neurons and modulate the mucus secretion in mouse olfactory organ.

Male pheromones and their reception by females are co-adapted to affect mating success in two subspecies of brown rats.

Pheromonal communication plays a key role in the sociosexual behavior of rodents. The coadaptation between pheromones and chemosensory systems has been well illustrated in insects but poorly investigated in rodents and other mammals. We aimed to investigate whether coadaptation between male pheromones and female reception might have occurred in brown rats Rattus norvegicus. We recently reported that major urinary protein (MUP) pheromones are associated with male mating success in a brown rat subspecies, R. n. humiliatus (Rnh). Here, we discovered that MUPs were less polymorphic and occurred at much lower concentrations in males of a parapatric subspecies, R. n. caraco (Rnc), than in Rnh males, and found no association between pheromones and paternity success. Moreover, the observation of Rnc males that experienced chronic dyadic encounters and established dominance–submission relationships revealed that the dominant males achieved greater mating success than the subordinate males, but their MUP levels did not differ by social status. These findings suggest that male mating success in Rnc rats is related to social rank rather than to pheromone levels and that low concentration of MUPs might not be a reliable signal for mate choice in Rnc rats, which is different from the findings obtained in Rnh rats. In addition, compared with Rnh females, Rnc females exhibited reduced expression of pheromone receptor genes, and a lower number of vomeronasal receptor neurons were activated by MUP pheromones, which imply that the female chemosensory reception of pheromones might be structurally and functionally coadapted with male pheromone signals in brown rats.

Main Olfactory and Vomeronasal Epithelium Are Differently Affected in Niemann-Pick Disease Type C1.

Introduction: Olfactory impairment is one of the earliest symptoms in neurodegenerative disorders that has also been documented in Niemann-Pick disease type C1 (NPC1). NPC1 is a very rare, neurovisceral lipid storage disorder, characterized by a deficiency of Npc1 gene function that leads to progressive neurodegeneration. Here, we compared the pathologic effect of defective Npc1 gene on the vomeronasal neuroepithelium (VNE) with that of the olfactory epithelium (OE) in an NPC1 mouse model. Methods: Proliferation in the VNE and OE was assessed by applying a bromodeoxyuridine (BrdU) protocol. We further compared the immunoreactivities of anti-olfactory marker protein (OMP), and the lysosomal marker cathepsin-D in both epithelia. To investigate if degenerative effects of both olfactory systems can be prevented or reversed, some animals were treated with a combination of miglustat/allopregnanolone/2-hydroxypropyl-cyclodextrin (HPβCD), or a monotherapy with HPβCD alone. Results: Using BrdU to label dividing cells of the VNE, we detected a proliferation increase of 215% ± 12% in Npc1−/− mice, and 270% ± 10% in combination- treated Npc1−/− animals. The monotherapy with HPβCD led to an increase of 261% ± 10.5% compared to sham-treated Npc1−/− mice. Similar to the OE, we assessed the high regenerative potential of vomeronasal progenitor cells. OMP reactivity in the VNE of Npc1−/− mice was not affected, in contrast to that observed in the OE. Concomitantly, cathepsin-D reactivity in the VNE was virtually absent. Conclusion: Vomeronasal receptor neurons are less susceptible against NPC1 pathology than olfactory receptor neurons. Compared to control mice, however, the VNE of Npc1−/− mice displays an increased neuroregenerative potential, indicating compensatory cell renewal.

Purinergic receptor-induced Ca2+ signaling in the neuroepithelium of the vomeronasal organ of larval Xenopus laevis

Purinergic signaling has considerable impact on the functioning of the nervous system, including the special senses. Purinergic receptors are expressed in various cell types in the retina, cochlea, taste buds, and the olfactory epithelium. The activation of these receptors by nucleotides, particularly adenosine-5′-triphosphate (ATP) and its breakdown products, has been shown to tune sensory information coding to control the homeostasis and to regulate the cell turnover in these organs. While the purinergic system of the retina, cochlea, and taste buds has been investigated in numerous studies, the available information about purinergic signaling in the olfactory system is rather limited. Using functional calcium imaging, we identified and characterized the purinergic receptors expressed in the vomeronasal organ of larval Xenopus laevis. ATP-evoked activity in supporting and basal cells was not dependent on extracellular Ca2+. Depletion of intracellular Ca2+ stores disrupted the responses in both cell types. In addition to ATP, supporting cells responded also to uridine-5′-triphosphate (UTP) and adenosine-5′-O-(3-thiotriphosphate) (ATPγS). The response profile of basal cells was considerably broader. In addition to ATP, they were activated by ADP, 2-MeSATP, 2-MeSADP, ATPγS, UTP, and UDP. Together, our findings suggest that supporting cells express P2Y2/P2Y4-like purinergic receptors and that basal cells express multiple P2Y receptors. In contrast, vomeronasal receptor neurons were not sensitive to nucleotides, suggesting that they do not express purinergic receptors. Our data provide the basis for further investigations of the physiological role of purinergic signaling in the vomeronasal organ and the olfactory system in general.

Expression and vesicular localization of mouse Trpml3 in stria vascularis, hair cells, and vomeronasal and olfactory receptor neurons

TRPML3 is a member of the mucolipin branch of the transient receptor potential cation channel family. A dominant missense mutation in Trpml3 (also known as Mcoln3) causes deafness and vestibular impairment characterized by stereocilia disorganization, hair cell loss, and endocochlear potential reduction. Both marginal cells of the stria vascularis and hair cells express Trpml3 mRNA. Here we used in situ hybridization, quantitative RT-qPCR, and immunohistochemistry with several antisera raised against TRPML3 to determine the expression and subcellular distribution of TRPML3 in the inner ear as well as in other sensory organs. We also use Trpml3 knockout tissues to distinguish TRPML3-specific from nonspecific immunoreactivities. We find that TRPML3 localizes to vesicles of hair cells and strial marginal cells but not to stereociliary ankle links or pillar cells, which nonspecifically react with two antisera raised against TRPML3. Upon cochlear maturation, TRPML3 protein is redistributed to perinuclear vesicles of strial marginal cells and is augmented in inner hair cells vs. outer hair cells. Mouse somatosensory neurons, retinal neurons, and taste receptor cells do not appear to express physiologically relevant levels of TRPML3. Finally, we found that vomeronasal and olfactory sensory receptor cells do express TRPML3 mRNA and protein, which localizes to vesicles in their somas and dendrites as well as at apical dendritic knobs.

Prion Shedding from Olfactory Neurons into Nasal Secretions

This study investigated the role of prion infection of the olfactory mucosa in the shedding of prion infectivity into nasal secretions. Prion infection with the HY strain of the transmissible mink encephalopathy (TME) agent resulted in a prominent infection of the olfactory bulb and the olfactory sensory epithelium including the olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs), whose axons comprise the two olfactory cranial nerves. A distinct glycoform of the disease-specific isoform of the prion protein, PrPSc, was found in the olfactory mucosa compared to the olfactory bulb, but the total amount of HY TME infectivity in the nasal turbinates was within 100-fold of the titer in the olfactory bulb. PrPSc co-localized with olfactory marker protein in the soma and dendrites of ORNs and VRNs and also with adenylyl cyclase III, which is present in the sensory cilia of ORNs that project into the lumen of the nasal airway. Nasal lavages from HY TME-infected hamsters contained prion titers as high as 103.9 median lethal doses per ml, which would be up to 500-fold more infectious in undiluted nasal fluids. These findings were confirmed using the rapid PrPSc amplification QuIC assay, indicating that nasal swabs have the potential to be used for prion diagnostics. These studies demonstrate that prion infection in the olfactory epithelium is likely due to retrograde spread from the olfactory bulb along the olfactory and vomeronasal axons to the soma, dendrites, and cilia of these peripheral neurons. Since prions can replicate to high levels in neurons, we propose that ORNs can release prion infectivity into nasal fluids. The continual turnover and replacement of mature ORNs throughout the adult lifespan may also contribute to prion shedding from the nasal passage and could play a role in transmission of natural prion diseases in domestic and free-ranging ruminants.

Heterogeneous Distribution of G Protein Alpha Subunits in the Main Olfactory and Vomeronasal Systems ofRhinella(Bufo)arenarumTadpoles

We evaluated the presence of G protein subtypes Galpha(o), Galpha(i2), and Galpha(olf) in the main olfactory system (MOS) and accessory or vomeronasal system (VNS) of Rhinella (Bufo) arenarum tadpoles, and here describe the fine structure of the sensory cells in the olfactory epithelium (OE) and vomeronasal organ (VNO). The OE shows olfactory receptor neurons (ORNs) with cilia in the apical surface, and the vomeronasal receptor neurons (VRNs) of the VNO are covered with microvilli. Immunohistochemistry detected the presence of at least two segregated populations of ORNs throughout the OE, coupled to Galpha(olf) and Galpha(o). An antiserum against Galpha(i2) was ineffective in staining the ORNs. In the VNO, Galpha(o) neurons stained strongly but lacked immunoreactivity to any other Galpha subunit in all larval stages analyzed. Western blot analyses and preabsorption experiments confirmed the specificity of the commercial antisera used. The functional significance of the heterogeneous G-protein distribution in R. arenarum tadpoles is not clear, but the study of G- protein distributions in various amphibian species is important, since this vertebrate group played a key role in the evolution of tetrapods. A more complete knowledge of the amphibian MOS and VNS would help to understand the functional organization and evolution of vertebrate chemosensory systems. This work demonstrates, for the first time, the existence of a segregated distribution of G-proteins in the OE of R. arenarum tadpoles.

A Novel Embryonic Nestin-Expressing Radial Glia-Like Progenitor Gives Rise to Zonally Restricted Olfactory and Vomeronasal Neurons

Persistent neurogenesis is maintained throughout development and adulthood in the mouse olfactory epithelium (OE). Despite this, the identity and origin of different embryonic OE progenitors, their spatiotemporal induction and contribution to patterning during development, has yet to be delineated. Here, we show that the embryonic OE contains a novel nestin-expressing radial glia-like progenitor (RGLP) that is not found in adult OE, which is antigenically distinct from embryonic CNS radial glia. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate "CNS-specific" nestin regulatory elements, and produce spatially restricted olfactory receptor neurons (ORNs) in zone 1 of the OE, and vomeronasal receptor neurons restricted to the VR1 zone. This dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, in which green fluorescent protein (GFP) is found in a subpopulation of GFP+Mash1+ neuronal progenitors, despite the fact that endogenous Nestin expression is found in RGLPs throughout the OE. Embryonic OE progenitors produce three biologically distinct colony subtypes in vitro, a subpopulation of which include nestin-expressing RGLPs during in vitro colony formation. When generated from Nestin-cre/ZEG mice, neurogenic colonies also produce GFP+Mash1+ progenitors and ORNs. We thus identify a novel neurogenic precursor, the RGLP of the OE and vomeronasal organ (VNO), and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during development of the OE and VNO.

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.

Expression of a vomeronasal receptor gene (V1r) and G protein α subunits in goat, Capra hircus, olfactory receptor neurons

Most mammals have two distinct olfactory epithelia, the olfactory epithelium (OE) and vomeronasal epithelium (VNE), containing, respectively, olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs). Olfactory receptors (ORs), which couple to G alpha olf, are generally expressed by ORNs, whereas two vomeronasal receptor families (V1rs and V2rs) coupled respectively to G alpha i2 and G alpha o, are expressed by VRNs. Previously, we reported that one goat V1rs (gV1ra1) is expressed by ORNs and VRNs. To investigate the characteristics of vomeronasal-receptor-expressing ORNs in mammals we performed double-label in situ hybridization for gV1ra1, G alpha i2, G alpha olf, olfactory marker protein (OMP), and growth association protein 43 (GAP43). Goat V1r-expressing ORNs are categorized into two types situated in different areas of the epithelium. The first type of V1r-expressing ORN coexpressed G alpha i2, but not OMP or GAP43. The second type of V1r-expressing ORN expresses G alpha olf and OMP, but not G alpha i2 or GAP43. These findings suggest that the two types of V1r-expressing ORN in goat OE function using different G protein alpha subunits for chemoreception.

Distribution of Notch1-expressing cells and proliferating cells in mouse vomeronasal organ

Vomeronasal receptor neurons (VRNs) proliferate and differentiate continuously in the vomeronasal organ (VNO) throughout life. In adult mice, new VRNs are generated mainly in the marginal region, located in the boundary region between sensory and nonsensory epithelia. The Notch signaling pathway is involved in differentiation in the developing nervous system. To understand the Notch signaling pathway involved in generating VRNs, we focused on the relationship between the expression pattern of Notch1 and the localization of proliferating cells in both developing and regenerating mice VNO, and examined the Notch signaling pathway involved in the development of VNO by in situ hybridization of Notch1 and immunocytochemistry of 5-bromo-2′-deoxyuridine. During embryonic and neonatal development, proliferating cells and Notch1-expressing (+) cells were observed evenly throughout VNO. A large number of proliferating cells and Notch1 (+) cells were observed in embryonic VNO, but gradually decreased during development. The localization of proliferating cells was similar to that of Notch1 (+) cells at each developmental stage. In adult VNO, there are a few proliferating cells and Notch1 (+) cells, which were only in the marginal region of VNO. Seven days after removal of the accessory olfactory bulb (AOB), VRNs proliferated throughout VNO. Although the number of Notch1 (+) cells also increased in VNO, the majority of these were concentrated in the dorsal region of VNO, suggesting that it has two types of differentiating cell. These results suggest that Notch1 plays a role in the differentiation of VRNs during development and regeneration of VRNs after removal of AOB.

The vomeronasal system and pheromones

The vomeronasal system and pheromones

Functional synapse formation between cultured rat accessory olfactory bulb neurons and vomeronasal pockets

To investigate the interaction between vomeronasal receptor neurons and accessory olfactory bulb neurons during pheromonal signal processing and specific synapse formation, partially dissociated rat vomeronasal receptor neurons were co-cultured with accessory olfactory bulb neurons. Between 7 and 14 days in co-culture, a few bundles of fibers from a spherical structure, termed the vomeronasal pocket, of cultured vomeronasal receptor neurons extended to the accessory olfactory bulb neurons. An optical recording of the intracellular Ca2+ concentration was used to monitor the synaptic activation of cultured accessory olfactory bulb neurons. Electrical stimulation of the vomeronasal pocket between 7 and 14 days in co-culture had no effects on most of the cultured neurons tested, although it occasionally evoked weak responses in a small number of neurons. In contrast, vomeronasal pocket stimulation after 21 days in co-culture evoked clear calcium transients in a substantial number of cultured accessory olfactory bulb neurons. These responses of accessory olfactory bulb neurons were reversibly suppressed by the application of 6-cyano-7-nitroquinoxaline-2,3-dione; the calcium transients disappeared in most of the neurons and were diminished in the others. The application of d-2-amino-5-phosphonopentanoic acid partially affected the calcium transients, but blocked spontaneous calcium increases, which were observed repeatedly in accessory olfactory bulb-alone cultures. The application of both 6-cyano-7-nitroquinoxaline-2,3-dione and d-2-amino-5-phosphonopentanoic acid completely blocked the evoked calcium transients. These results suggest that functional glutamatergic synapses between vomeronasal receptor neurons and accessory olfactory bulb neurons were formed at around 21 days in co-culture.

Ultrastructural localization of α-galactose-containing glycoconjugates in the rat vomeronasal organ

Binding sites of Griffonia simplicifolia I-B4 isolectin (GS-I-B4), which recognizes terminal alpha-galactose residues of glycoconjugates, were examined in the juxtaluminal region of the rat vomeronasal sensory epithelium and its associated glands of the vomeronasal organ, using a lectin cytochemical technique. Lowicryl K4M-embedded ultra-thin sections, which were treated successively with biotinylated GS-I-B4 and streptavidin-conjugated 10 nm colloidal gold particles, were observed under a transmission electron microscope. Colloidal gold particles, which reflect the presence of terminal alpha-galactose-containing glycoconjugates, were present in vomeronasal receptor neurons in the sensory epithelium and secretory granules of acinar cells of associated glands of the epithelium. Quantitative analysis demonstrated that the density of colloidal gold particles associated with sensory cell microvilli that projected from dendritic endings of vomeronasal neurons was considerably higher than that of microvilli that projected from neighboring sustentacular cells. The same was true for the apical cytoplasms of these cells just below the microvilli. These results suggest that of the sensory microvilli and dendritic endings contained a much larger amount of the alpha-galactose-containing glycoconjugates, compared with those in sustentacular microvilli. Further, biochemical analyses demonstrated several vomeronasal organ-specific glycoproteins with terminal alpha-galactose.

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.

The Roles of Brain-derived Neurotrophic Factor in the Development of Nasal Chemoreceptor Neurons

Brain-derived neurotrophic factor (BDNF) is one of the neurotrophins, and known to facilitate differentiation, growth and maturation of neurons. BDNF binds to the high-affinity receptor, tyrosine kinase receptor B (TrkB) to initiate signal transduction (Korsching, 1993; Lindsay et al., 1994). Olfactory and vomeronasal receptor neurons (ORNs and VRNs, respectively) are chemoreceptors located in the nasal cavity of most mammalian species (Graziadei, 1977). In the olfactory epithelium (OE) lining the olfactory mucosa (OM), progenitor cells differentiate into immature and mature ORNs; a single dendrite extending from the apical pole of each soma reaches the surface of the OE to form a dendritic ending, and a single axon from the base of the soma runs downward to pass through the basement membrane of the OE to reach the brain. Similar bipolar neurons are also contained in the vomeronasal sensory epithelium (VSE) of the vomeronasal organ (VNO), and designated as VRNs or vomeronasal sensory cells/ neurons (Graziadei, 1977; Takami, 2002). Although several neurotrophic factors including neurotrophins are thought to be involved in the development, maturation, and regeneration of ORNs (Carter and Roskams, 2002; Schwob, 2002), the functional roles of BDNF remain to be understood. In the case of the VRNs, we were the first to report the distribution of BDNF and TrkB at both domestic (Takami et al., 2001) and international (Takami and Nishiyama, 1997b) meetings. In this paper, we present summary of our recent studies concerning BDNF and TrkB in the rat OM and VNO. Our overall research goal is to clarify functional roles of BDNF in the differentiation and maturation of ORNs and VRNs.

Functional synapse formation between cultured rat accessory olfactory bulb neurons and vomeronasal pockets

To investigate the interaction between vomeronasal receptor neurons and accessory olfactory bulb neurons during pheromonal signal processing and specific synapse formation, partially dissociated rat vomeronasal receptor neurons were co-cultured with accessory olfactory bulb neurons. Between 7 and 14 days in co-culture, a few bundles of fibers from a spherical structure, termed the vomeronasal pocket, of cultured vomeronasal receptor neurons extended to the accessory olfactory bulb neurons. An optical recording of the intracellular Ca(2+) concentration was used to monitor the synaptic activation of cultured accessory olfactory bulb neurons. Electrical stimulation of the vomeronasal pocket between 7 and 14 days in co-culture had no effects on most of the cultured neurons tested, although it occasionally evoked weak responses in a small number of neurons. In contrast, vomeronasal pocket stimulation after 21 days in co-culture evoked clear calcium transients in a substantial number of cultured accessory olfactory bulb neurons. These responses of accessory olfactory bulb neurons were reversibly suppressed by the application of 6-cyano-7-nitroquinoxaline-2,3-dione; the calcium transients disappeared in most of the neurons and were diminished in the others. The application of d-2-amino-5-phosphonopentanoic acid partially affected the calcium transients, but blocked spontaneous calcium increases, which were observed repeatedly in accessory olfactory bulb-alone cultures. The application of both 6-cyano-7-nitroquinoxaline-2,3-dione and d-2-amino-5-phosphonopentanoic acid completely blocked the evoked calcium transients. These results suggest that functional glutamatergic synapses between vomeronasal receptor neurons and accessory olfactory bulb neurons were formed at around 21 days in co-culture.

Selective ablation of olfactory receptor neurons without functional impairment of vomeronasal receptor neurons in OMP-ntr transgenic mice.

This study used transgenic mice, in which expression of a bacterial nitroreductase (ntr) gene was linked to the expression of olfactory marker protein (OMP). The nitroreductase enzyme is thus expressed in mature chemosensory neurons of these OMP-ntr transgenic mice, and converts the pro-drug CB1954 to a cytotoxic form, specifically killing these neurons. Systemic injections of the pro-drug led to the ablation of receptor neurons in both the main olfactory and vomeronasal epithelia. Due to the anatomical separation of the epithelia, however, when the pro-drug was administered by intranasal infusion only the receptors of the main olfactory epithelium were destroyed. This procedure resulted in a profound deficit in olfactory investigation and discrimination in a habituation-dishabituation test, whereas the pregnancy blocking effect of male pheromones, which is mediated via the vomeronasal system was unaffected. OMP-ntr mice receiving intranasal infusion of pro-drug had not recovered any significant main olfactory function at 24 days following treatment. This novel technique could potentially be applied to selectively ablate olfactory receptor neurons expressing a particular olfactory receptor by linking its expression to that of the nitroreductase enzyme.

Arachidonic Acid Plays a Role in Rat Vomeronasal Signal Transduction

Sensory neurons of the vomeronasal organ (VNO) detect volatile chemicals that are released by conspecific animals and convey information about social and reproductive behavior. The signal transduction pathway in vomeronasal receptor neurons (VRNs) is not known in detail, but is believed to be distinct from that of the sensory neurons of the main olfactory system. Many of the identified olfactory transduction components are not expressed by VRNs. Using Ca2+ imaging and electrophysiological recordings, we investigated the signal transduction pathway of urine perception and the possible role of polyunsaturated fatty acids (PUFAs) as intracellular messengers in freshly dissociated rat VNO neurons. We found that application of urine induced a transient increase in intracellular Ca2+ that was dependent on the activity of phospholipase C and diacylglycerol (DAG) lipase. The Ca2+ transient was not dependent on depletion of intracellular Ca2+ stores but was dependent on the presence of extracellular Ca2+. Furthermore, the urine response was not sensitive to modulators of adenylate cyclase and inhibitors of inositol 1,4,5-trisphosphate receptors. Application of PUFAs (linolenic acid and arachidonic acid, synthesized in living cells from DAG) also elicited Ca2+ transients in fura 2 measurements and inward currents in whole-cell voltage-clamp recordings. Pharmacological inhibition of lipoxygenase and cyclooxygenase induced a transient increase in intracellular Ca2+, possibly by increasing the endogenous level of PUFAs, leading to activation of transduction channels. These data provide evidence for a role of PUFAs in rat vomeronasal signal transduction.