Neuropeptide Y enhances olfactory mucosa responses to odorant in hungry rats.

Neuropeptide Y (NPY) plays an important role in regulating appetite and hunger in vertebrates. In the hypothalamus, NPY stimulates food intake under the control of the nutritional status. Previous studies have shown the presence of NPY and receptors in rodent olfactory system, and suggested a neuroproliferative role. Interestingly, NPY was also shown to directly modulate olfactory responses evoked by a food-related odorant in hungry axolotls. We have recently demonstrated that another nutritional cue, insulin, modulates the odorant responses of the rat olfactory mucosa (OM). Therefore, the aim of the present study was to investigate the potential effect of NPY on rat OM responses to odorants, in relation to the animal's nutritional state. We measured the potential NPY modulation of OM responses to odorant, using electro-olfactogram (EOG) recordings, in fed and fasted adult rats. NPY application significantly and transiently increased EOG amplitudes in fasted but not in fed rats. The effects of specific NPY-receptor agonists were similarly quantified, showing that NPY operated mainly through Y1 receptors. These receptors appeared as heterogeneously expressed by olfactory neurons in the OM, and western blot analysis showed that they were overexpressed in fasted rats. These data provide the first evidence that NPY modulates the initial events of odorant detection in the rat OM. Because this modulation depends on the nutritional status of the animal, and is ascribed to NPY, the most potent orexigenic peptide in the central nervous system, it evidences a strong supplementary physiological link between olfaction and nutritional processes.

Leptin-sensitive OBP-expressing mucous cells in rat olfactory epithelium: a novel target for olfaction-nutrition crosstalk?

Although odorant-binding proteins (OBP) are one of the most abundant classes of proteins in the mammalian olfactory mucus, they have only recently been ascribed a functional role in the detection of odorants by olfactory neurons. Among the three OBPs described in the rat, OBP-1f is mainly secreted by the lateral nasal glands (LNG) and Bowman's glands, and its expression is transcriptionally regulated by food deprivation in the olfactory mucosa, but not in LNG. Therefore, mucus composition might be locally regulated by hormones or molecules relevant to nutritional status. Our aim has been to investigate the mechanisms of such physiological regulation at the cellular level, through both the examination of OBP-1f synthesis sites in the olfactory mucosa and their putative regulation by leptin, a locally acting satiety hormone. Immunohistochemical observations have allowed the identification of a novel population of OBP-1f-secreting cells displaying morphological and functional characteristics similar to those of epithelial mucous cells. Ultrastructural analyses by both transmission and scanning electron microscopy has enabled a more complete cytoarchitectural characterization of these specialized olfactory mucous cells in their tissue environment. These globular cells are localized in discrete zones of the olfactory epithelium, mainly in the fourth turbinate, and are often scattered from the basal to the apical surface of the epithelium. They contain numerous small droplets of mucosubstances. Using an in-vitro-derived model of olfactory mucosa primary culture, we have been able to demonstrate that leptin increases the production of mucus by these cells, so that they constitute potential targets for the physiological modulation of mucus composition by nutritional cues.

On a chip demonstration of a functional role for Odorant Binding Protein in the preservation of olfactory receptor activity at high odorant concentration.

The molecular mechanisms underlying odorant detection have been investigated using the chip based SPR technique by focusing on the dynamic interactions between transmembrane Olfactory Receptor OR1740, odorant ligands and soluble Odorant-Binding Protein (OBP-1F). The OR1740 present in the lipid bilayer of nanosomes derived from transformed yeasts specifically bound OBP-1F. The receptor preferential odorant ligand helional released bound OBP-1F from the OR-OBP complex, while unrelated odorants failed to do so. OBP-1F modified the functional OR1740 dose-response to helional, from a bell-shaped to a saturation curve, thus preserving OR activity at high ligand concentration. This unravels an active role for OBPs in olfaction, in addition to passive transport or a scavenger role. This sensorchip technology was applied to assessing native OBP-1F in a biological sample: rat olfactory mucus also displayed significant binding to OR1740 nanosomes, and the addition of helional yielded the dissociation of mucus OBP from the receptor.

Gold surface functionalization and patterning for specific immobilization of olfactory receptors carried by nanosomes.

There is substantial interest in engineering solid supports to achieve functional immobilization of membrane receptors both for investigation of their biological function and for the development of novel biosensors. Three simple and practical strategies for immobilization of a human olfactory receptor carried by nanosomes are presented. The basis of the functionalization of solid gold surfaces is a self-assembled monolayer (SAM) containing biotinyl groups. Biotinyl groups are subsequently used to attach neutravidin and then biotinylated monoclonal antibody directed against the receptor to allow its specific grafting. Surface plasmon resonance technique is implemented for real-time monitoring of step-by-step surface functionalization and, in addition, for testing the functional response of immobilized olfactory receptors. We show that OR1740 is functional when immobilized via a tag attached to its C-terminus, but not via its N-terminus. Finally, we demonstrate that gold surfaces can be patterned by the SAMs tested using microcontact printing. AFM images of immobilized nanosomes onto a patterned surface suggest that small nanosomes flatten and fuse into larger vesicles but do not merge into a continuous layer. The whole study emphasizes the outstanding performances of the BAT/PEGAT SAM, which could be useful for developing on-a-chip sensor formats for membrane receptor investigations and use.

Study of orexins signal transduction pathways in rat olfactory mucosa and in olfactory sensory neurons-derived cell line Odora: multiple orexin signalling pathways.

Orexins A and B (OxA and OxB) are multifunctional neuropeptides implicated in the regulation of energy metabolism, wakefulness but also in a broad range of motivated behaviours. They signal through two G-protein-coupled receptors: orexin receptor 1 and 2 (Ox1R and Ox2R). The orexins and their receptors are present at all levels of the rat olfactory system: epithelium, bulb, piriform cortex but their signalling mechanisms remain unknown. We have studied orexins signal transduction pathways in the rat olfactory mucosa (OM) and in the Odora cell line derived from olfactory sensory neurons and heterologously expressing Ox1R or Ox2R. We have demonstrated by western blot and RT-PCR that multiple components of adenylyl cyclase (AC) and phospholipase C (PLC) signalling pathways were identical in OM and Odora cells. OxA and OxB induced a weak increase in IP3 in OM; they induced a significant rise in cAMP and IP3 in Odora transfected cells, suggesting the activation of AC and PLC pathways. Both OxA and OxB induced intracellular calcium elevation and transient activation of MAP kinases (ERK42/44) in Odora/Ox1R and Odora/Ox2R cells. These results suggest the existence of multiple orexins signalling pathways in Odora cells and probably in OM, corresponding to different possible roles of these peptides.

A novel detection strategy for odorant molecules based on controlled bioengineering of rat olfactory receptor I7.

In this study, we report a dose-dependent detection of odorant molecules in solution by rat olfactory receptor I7 (OR I7) in its membrane fraction. The OR I7 is immobilized on a gold electrode by multilayer bioengineering based on a mixed self-assembled monolayer and biotin/avidin system, which allows for a well-controlled immobilization of the bioreceptor within its lipid environment. The odorant detection is electronically performed in a quantitative manner by electrochemical impedance spectroscopy (EIS) measurements on samples and controls.

Leptin and its receptors are present in the rat olfactory mucosa and modulated by the nutritional status.

Leptin is an adipocyte-derived cytokine that regulates body weight mainly via the long form of the leptin receptor (Ob-Rb). Leptin and its receptors are expressed in several tissues, suggesting that leptin might also be effective peripherally. We hypothesized that, as shown in taste cells, leptin and its receptors isoforms (Ob-Rs) could be present in the rat olfactory mucosa (OM). Using RT-PCR, light and electron microscopy immunohistochemistry (ICC), we found that different isoforms of the receptor were expressed in OM and localized in sustentacular cells and in a subpopulation of maturating neurons; in addition, immunoreactivity was also present in differentiated neurons and enriched at the cilia membranes, where the odorants bind to their receptors. Moreover, using RT-PCR, ICC and RIA measurements, we showed that leptin is synthesized locally in the olfactory mucosa. In addition, we demonstrate that fasting causes a significant enhanced transcription of both leptin and Ob-Rs in rat OM by quantitative RT-PCR data. Altogether, these results strongly suggested that leptin, acting as an endocrine or a paracrine factor, could be an important regulator of olfactory function, as a neuromodulator of the olfactory message in cilia of mature olfactory receptors neurons (ORN), but also for the homeostasis of this complex tissue, acting on differentiating neurons and on sustentacular cells.

Quantitative assessment of olfactory receptors activity in immobilized nanosomes: a novel concept for bioelectronic nose.

We describe how mammalian olfactory receptors (ORs) could be used as sensing elements of highly specific and sensitive bioelectronic noses. An OR and an appropriate G(alpha) protein were co-expressed in Saccharomyces cerevisiae cells from which membrane nanosomes were prepared, and immobilized on a sensor chip. By Surface Plasmon Resonance, we were able to quantitatively evaluate OR stimulation by an odorant, and G protein activation. We demonstrate that ORs in nanosomes discriminate between odorant ligands and unrelated odorants, as in whole cells. This assay also provides the possibility for quantitative assessment of the coupling efficiency of the OR with different G(alpha) subunits, without the interference of the cellular transduction pathway. Our findings will be useful to develop a new generation of electronic noses for detection and discrimination of volatile compounds, particularly amenable to micro- and nano-sensor formats.

Immobilization of rhodopsin on a self-assembled multilayer and its specific detection by electrochemical impedance spectroscopy.

Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.

Orexin A modulates mitral cell activity in the rat olfactory bulb: patch-clamp study on slices and immunocytochemical localization of orexin receptors.

Orexin A and B are involved in feeding behaviors, and recently fibers containing these peptides were found in the rat olfactory bulb. These fibers, which originate from the lateral and posterior hypothalamus and the perifornical area, are distributed in the glomerular, mitral cell, and granule cell layers. Orexin receptors are mainly expressed by mitral cells. In the present study, RT-PCR experiments were done to determine orexin receptor expression during the early postnatal life of rats, and immunocytochemical experiments were performed to further clarify the structural and ultrastructural localization of orexin receptors in the olfactory bulb. Furthermore, a functional electrophysiological approach examined the action of orexin A on mitral cell excitability and spontaneous activity using in vitro patch-clamp techniques. RT-PCR results show that mRNA of the two type receptors, type 1 orexin receptors and type 2 orexin receptors, are expressed in the olfactory bulb of rat from 10 d to the adult stage. At the same ages, immunocytochemical data show that orexin 1 receptors are localized in the cell bodies of periglomerular, mitral/tufted, and granule cells. Immunoreactivity was also demonstrated in mitral/tufted cell dendrites arborizing in the glomerulus and mitral/tufted and granule cell processes running in the external plexiform layer. Functionally, orexin A produced either a direct, tetrodotoxin-insensitive depolarization in one group of mitral cells (7%), or, in another group (30%), an indirect, tetrodotoxin-sensitive hyperpolarization. Both actions were mediated by type 1 orexin receptors because the response was antagonized by SB-334867-A, a selective antagonist. Mitral cell recordings performed under bicuculline [gamma-aminobutyric acid (GABA)A receptor antagonist], indicate that the orexin-induced indirect hyperpolarization was partly mediated through GABA(A) receptors. Because granule cells and periglomerular cells express orexin receptors and are GABAergic cells, they could be both involved in this hyperpolarization. Other mechanisms, which could support an indirect hyperpolarization of mitral cells through dopamine interneuron solicitation, are proposed. Our results provide data that should allow us to better understand neural communication and regulation mechanisms between the hypothalamic feeding centers and the olfactory bulb.

Immobilization of native membrane-bound rhodopsin on biosensor surfaces.

In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.

Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening.

The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system. The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands. The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional. Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms.

Anatomical and functional evidence for a role of arginine-vasopressin (AVP) in rat olfactory epithelium cells.

The olfactory epithelium (OE) is composed of olfactory sensory neurons (OSNs) and sustentacular cells; it lies in the nasal cavity where it is protected by a thin mucus layer. The finely regulated composition of this mucus provides OSN with a suitable ionic environment. To maintain the functional integrity of the epithelium despite permanent physical, chemical and microbial aggressions, both OSNs and surrounding sustentacular cells are continuously renewed from globose basal cells. Moreover, the sense of smell is involved in so numerous behaviours (feeding, reproduction, etc.) that it has to cross-talk with the endocrine and neuroendocrine systems. Thus, besides its sensory function, the olfactory epithelium is thought to undergo a lot of complex regulatory processes. We therefore studied the effects of various neuropeptides on primary cultures of Sprague-Dawley rat olfactory epithelium cells. We found that arginine-vasopressin (AVP) triggered a robust, dose-dependent calcium increase in these cells. The cell response was essentially ascribed to the V1a AVP receptor, whose presence was confirmed by RT-PCR and immunolabelling. In the culture, V1a but not V1b receptors were present, mainly localized in neurons. In the epithelium, both subtypes were found differentially distributed. V1a-R were localized mainly in globose basal cells and at the apical side of the epithelium, in the area of the dendritic knobs of OSNs. V1b-R were strongly associated with Bowman's gland cells and globose basal cells. These localizations suggested potential multifaceted roles of a hormone, AVP, in the olfactory epithelium.

hnRNP A1 and the SR proteins ASF/SF2 and SC35 have antagonistic functions in splicing of beta-tropomyosin exon 6B.

Mutually exclusive splicing of exons 6A and 6B from the chicken beta-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them.

Engineered yeasts as reporter systems for odorant detection.

The functional expression of olfactory receptors (ORs) is a primary requirement to utilize olfactory detection systems. We have taken advantage of the functional similarities between signal transduction cascades in the budding yeast Saccharomyces cerevisiae and mammalian cells. The yeast pheromone response pathway has been adapted to allow ligand-dependent signaling of heterologous expressed G-protein coupled receptors (GPCRs) via mammalian or chimeric yeast/mammalian Galpha proteins. Two different strategies are reported here which offer a positive screen for functional pairs. The OR and Galpha protein are introduced into the modified yeast cells such that they hijack the pheromone response pathway usually resulting in cell cycle arrest. The first strategy utilizes ligand-induced expression of a FUS1-HIS3 reporter gene to permit growth on a selective medium lacking histidine; the second to induce ligand-dependent expression of a FUSI-Hph reporter gene, conferring resistance to hygromycin. Validation of the systems was performed using the rat 17 receptor response to a range of aldehyde odorants previously characterized as functional ligands. Of these only heptanal produced a positive growth response in the concentration range 5 x 10(-8) to 5 x 10(-6) M. Induction conditions appear to be critical for functional expression, and the solvents of odorants have a toxic effect for the highest odorant concentrations. The preference of rat 17 receptor for the ligand heptanal in yeast has to be compared to concurrent results obtained with mammalian expression systems.

Ligand-specific dose-response of heterologously expressed olfactory receptors.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.

Localization of orexins and their receptors in the rat olfactory system: possible modulation of olfactory perception by a neuropeptide synthetized centrally or locally.

Orexin-A and -B, also known as hypocretins, are two neuropeptides acting on feeding and sleep. They are specific ligands for two different receptors belonging to the G-protein coupled receptors family. Orexin fibers and orexin receptor neurons have been previously described in the forebrain olfactory system. Using immunocytochemistry, we showed that both orexin-A and -B as well as their receptors were present at different levels of the olfactory system, from the nasal mucosa to nuclei of the amygdala. A punctuated staining for orexins and their receptors was detected at the apical part of the olfactory epithelium; in the lamina propria of the mucosa, the staining was localized around olfactory nerves. At the ultrastructural level, olfactory neurons and supporting cells were found immunoreactive for orexins and their receptors. The labeling was localized in dendritic knobs and cilia of neurons, in the apical part and microvilli of supporting cells. The finding of immunolabeled cisternae of reticulum strongly suggests a local synthesis of both peptides and receptors, confirmed by RT-PCR experiments. In forebrain and amygdala regions, we detected numerous orexin fibers. Orexin receptors were present in mitral-tufted cells of the bulb and in many neuronal perikarya in the anterior olfactory nuclei, piriform cortex and amygdala nuclei. Altogether, these results show that orexins and their receptors are present at all levels of the olfactory system, from cilia where odors bind to their receptors to central regions where integration of olfactory signals occurs. They suggest a possible modulation of olfactory perception by these neuropeptides.

Rescue of intracellularly trapped lutropin receptor exodomain by endodomain and reconstitution of a functional membrane receptor: interaction between exo- and endodomains.

The lutropin receptor consists of an extracellular N-terminal half and a membrane-associated C-terminal half. hCG initially binds the exodomain with a high affinity and the resulting complex is thought to interact with the endodomain through a secondary contact generating a hormonal signal. Therefore, the exodomain and endodomain are likely to associate directly or indirectly with each other, but lack of fruitful materials and technology has hampered knowledge about their physical relationship and contact sites. In this work, we engineered a double-recombinant (separate exodomain and endodomain) baculovirus system successfully expressing on the surface of insect cells high levels of split LH receptor, binding the hormone with high affinity and inducing cAMP synthesis. In contrast, the exodomain and endodomain expressed separately were mostly trapped in cells. Our data indicate that the exodomain and endodomain are disulfide linked in the split receptor. When the disulfide links were reduced, the split receptor still induced cAMP up to 60%, which raises the intriguing possibility of a residual induction activity of the endodomain in the absence of high-affinity ligand binding. Our results also underscore that the targeting and transport of the LH receptor to plasma membrane require both domains, whereas each domain is independently sufficient for folding. The expression level of functional lutropin receptors is the highest ever reported. Our system may also be useful for future studies requiring a high amount of soluble secreted exodomain.