Characteristics of odorant elicited calcium fluxes in acutely-isolated chick olfactory neurons

To understand avian olfaction, it is important to characterize the peripheral olfactory system of a representative bird species. This study determined the functional properties of olfactory receptor neurons of the chicken olfactory epithelium. Individual neurons were acutely isolated from embryonic day-18 to newborn chicks by dissection and enzymatic dissociation. We tested single olfactory neurons with behaviorally relevant odorant mixtures and measured their responses using ratiometric calcium imaging; techniques used in this study were identical to those used in other studies of olfaction in other vertebrate species. Chick olfactory neurons displayed properties similar to those found in other vertebrates: they responded to odorant stimuli with either decreases or increases in intracellular calcium, calcium increases were mediated by a calcium influx, and responses were reversibly inhibited by 100 M L–cis–diltiazem, 1 mM Neomycin, and 20 M U73122, which are biochemical inhibitors of second messenger signaling. In addition, some cells showed a complex pattern of responses, with different odorant mixtures eliciting increases or decreases in calcium in the same cell. It appears that there are common features of odorant signaling shared by a variety of vertebrate species, as well as features that may be peculiar to chickens.     

Odorant receptors directly activate phospholipase C/inositol-1,4,5-trisphosphate coupled to calcium influx in Odora cells

Mechanisms by which odorants activate signaling pathways in addition to cAMP are hard to evaluate in heterogeneous mixtures of primary olfactory neurons. We used single cell calcium imaging to analyze the response to odorant through odorant receptor (OR) U131 in the olfactory epithelial cell line Odora (Murrell and Hunter 1999), a model system with endogenous olfactory signaling pathways. Because adenylyl cyclase levels are low, agents activating cAMP formation do not elevate calcium, thus unmasking independent signaling mediated by OR via phospholipase C (PLC), inositol-1,4,5-trisphosphate (IP(3)), and its receptor. Unexpectedly, we found that extracellular calcium is required for odor-induced calcium elevation without the release of intracellular calcium, even though the latter pathway is intact and can be stimulated by ATP. Relevant signaling components of the PLC pathway and G protein isoforms are identified by western blot in Odora cells as well as in olfactory sensory neurons (OSNs), where they are localized to the ciliary zone or cell bodies and axons of OSNs by immunohistochemistry. Biotinylation studies establish that IP(3) receptors type 2 and 3 are at the cell surface in Odora cells. Thus, individual ORs are capable of elevating calcium through pathways not directly mediated by cAMP and this may provide another avenue for odorant signaling in the olfactory system.     

Olfactory receptor neuron profiling using sandalwood odorants

The mammalian olfactory system can discriminate between volatile molecules with subtle differences in their molecular structures. Efforts in synthetic chemistry have delivered a myriad of smelling compounds of different qualities as well as many molecules with very similar olfactive properties. One important class of molecules in the fragrance industry are sandalwood odorants. Sandalwood oil and four synthetic sandalwood molecules were selected to study the activation profile of endogenous olfactory receptors when exposed to compounds from the same odorant family. Dissociated rat olfactory receptor neurons were exposed to the sandalwood molecules and the receptor activation studied by monitoring fluxes in the internal calcium concentration. Olfactory receptor neurons were identified that were specifically stimulated by sandalwood compounds. These neurons expressed olfactory receptors that can discriminate between sandalwood odorants with slight differences in their molecular structures. This is the first study in which an important class of perfume compounds was analyzed for its ability to activate endogenous olfactory receptors in olfactory receptor neurons.     

Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction

Fruit flies are attracted by a diversity of odors that signal the presence of food, potential mates, or attractive egg-laying sites. Most Drosophila olfactory neurons express two types of odorant receptor genes: Or83b, a broadly expressed receptor of unknown function, and one or more members of a family of 61 selectively expressed receptors. While the conventional odorant receptors are highly divergent, Or83b is remarkably conserved between insect species. Two models could account for Or83b function: it could interact with specific odor stimuli independent of conventional odorant receptors, or it could act in concert with these receptors to mediate responses to all odors. Our results support the second model. Dendritic localization of conventional odorant receptors is abolished in Or83b mutants. Consistent with this cellular defect, the Or83b mutation disrupts behavioral and electrophysiological responses to many odorants. Or83b therefore encodes an atypical odorant receptor that plays an essential general role in olfaction.     

Olfactory receptor antagonism between odorants

The detection of thousands of volatile odorants is mediated by several hundreds of different G protein-coupled olfactory receptors (ORs). The main strategy in encoding odorant identities is a combinatorial receptor code scheme in that different odorants are recognized by different sets of ORs. Despite increasing information on agonist-OR combinations, little is known about the antagonism of ORs in the mammalian olfactory system. Here we show that odorants inhibit odorant responses of OR(s), evidence of antagonism between odorants at the receptor level. The antagonism was demonstrated in a heterologous OR-expression system and in single olfactory neurons that expressed a given OR, and was also visualized at the level of the olfactory epithelium. Dual functions of odorants as an agonist and an antagonist to ORs indicate a new aspect in the receptor code determination for odorant mixtures that often give rise to novel perceptual qualities that are not present in each component. The current study also provides insight into strategies to modulate perceived odorant quality.     

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.     

Ion channels from chemosensory olfactory neurons

The olfactory epithelium has the ability to respond to a large number of volatile compounds of small molecular weight. Ultimately, such a property lies on a specialized type of neuron, the olfactory receptor cell. In the presence of odorants, the olfactory receptor neuron responds with action potentials whose frequency depends on odorant concentration. The primary events in the process of olfactory transduction are thought to occur at the cilia of olfactory receptor neurons and involve the binding of odorants to receptor molecules followed by the opening of ion channels. A crucial step in understanding olfactory transduction requires identifying the mechanisms that regulate the electrical activity of olfactory cells. In the last couple of years, patch-clamp recording from isolated olfactory cells and reconstitution of olfactory membranes in planar lipid bilayers have begun to shed light on some of these mechanisms. Although the information emerging from such studies is still preliminary, there are already well-defined hypotheses on the molecular events that might underlie the primary events in olfactory transduction. Currently, attention is being focused on the notions that second messengers might be involved in the activation of ion channels in olfactory cilia, and that odorant binding to a receptor molecule might lead directly to the gating of ion channels in chemosensory olfactory membranes. The coming years promise to be exciting ones in the field of olfactory transduction. We have now the necessary tools to be able to confront hypotheses and experimental facts.     

Imaging ensemble activity in arthropod olfactory receptor neurons in situ

We show that lobster olfactory receptor neurons (ORNs), much like their vertebrate counterparts, generate a transient elevation of intracellular calcium (Ca(i)) in response to odorant activation that can be used to monitor ensemble ORN activity. This is done in antennal slice preparation in situ maintaining the polarity of the cells and the normal micro-environment of the olfactory cilia. The Ca(i) signal is ligand-specific and increases in a dose-dependent manner in response to odorant stimulation. Saturating stimulation elicits a robust increase of up to 1 μM free Ca(i) within 1-2s of stimulation. The odor-induced Ca(i) response closely follows the discharge pattern of extracellular spikes elicited by odorant application, with the maximal rise in Ca(i) matching the peak of the spike generation. The Ca(i) signal can be used to track neuronal activity in a functional subpopulation of rhythmically active ORNs and discriminate it from that of neighboring tonically active ORNs. Being able to record from many ORNs simultaneously over an extended period of time not only allows more accurate estimates of neuronal population activity but also dramatically improves the ability to identify potential new functional subpopulations of ORNs, especially those with more subtle differences in responsiveness, ligand specificity, and/or transduction mechanisms.     

The role of the coreceptor Orco in insect olfactory transduction

Insects sense odorants with specialized odorant receptors (ORs). Each antennal olfactory receptor neuron expresses one OR with an odorant binding site together with a conserved coreceptor called Orco which does not bind odorants. Orco is necessary for localization of ORs to dendritic membranes and, thus, is essential for odorant detection. It forms a spontaneously opening cation channel, activated via phosphorylation by protein kinase C. Thereafter, Orco is also activated via cyclic adenosine monophosphate (cAMP). Orco forms homo—as well as heteromers with ORs with unknown stoichiometry. Contradictory publications suggest different mechanisms of olfactory transduction. On the one hand, evidence accumulates for the employment of more than one G protein-coupled olfactory transduction cascade in different insects. On the other hand, results from other studies suggest that the OR–Orco complex functions as an odorant-gated cation channel mediating ionotropic signal transduction. This review analyzes conflicting hypotheses concerning the role of Orco in insect olfactory transduction. In conclusion, in situ studies in hawkmoths falsify the hypothesis that Orco underlies odorant-induced ionotropic signal transduction in all insect species. Instead, Orco forms a metabotropically gated, slow cation channel which controls odorant response threshold and kinetics of the sensory neuron.     

Olfactory transduction: cross-talk between second-messenger systems

Chemosensory cilia of olfactory receptor neurons contain an adenylate cyclase which is stimulated by high concentrations of odorants. Cyclic AMP produced by this enzyme has been proposed to act as second messenger in olfactory transduction. Here we report that olfactory cilia contain calmodulin and that calmodulin potently activates olfactory adenylate cyclase by a mechanism additive to and independent from direct stimulation by odorants. Activation by calmodulin is calcium dependent and enhanced by GTP. Thus, olfactory transduction may involve a second-messenger cascade in which an odorant-induced increase in intracellular calcium concentration leads to activation of adenylate cyclase by calmodulin.     

Differential interactions of sex pheromone and plant odour in the olfactory pathway of a male moth

Most animals rely on olfaction to find sexual partners, food or a habitat. The olfactory system faces the challenge of extracting meaningful information from a noisy odorous environment. In most moth species, males respond to sex pheromone emitted by females in an environment with abundant plant volatiles. Plant odours could either facilitate the localization of females (females calling on host plants), mask the female pheromone or they could be neutral without any effect on the pheromone. Here we studied how mixtures of a behaviourally-attractive floral odour, heptanal, and the sex pheromone are encoded at different levels of the olfactory pathway in males of the noctuid moth Agrotis ipsilon. In addition, we asked how interactions between the two odorants change as a function of the males' mating status. We investigated mixture detection in both the pheromone-specific and in the general odorant pathway. We used a) recordings from individual sensilla to study responses of olfactory receptor neurons, b) in vivo calcium imaging with a bath-applied dye to characterize the global input response in the primary olfactory centre, the antennal lobe and c) intracellular recordings of antennal lobe output neurons, projection neurons, in virgin and newly-mated males. Our results show that heptanal reduces pheromone sensitivity at the peripheral and central olfactory level independently of the mating status. Contrarily, heptanal-responding olfactory receptor neurons are not influenced by pheromone in a mixture, although some post-mating modulation occurs at the input of the sexually isomorphic ordinary glomeruli, where general odours are processed within the antennal lobe. The results are discussed in the context of mate localization.     

Specificity of odorant-evoked inhibition in lobster olfactory receptor neurons

Lobster olfactory receptor neurons, like those of many animals, use two modes of olfactory signaling, excitation and inhibition to code olfactory information. Inhibition appears to act through two distinct ionic mechanisms. Here we show that neither ionic mechanism is odor-specific, providing further support for the emerging understanding that there are no inhibitory odorants per se, but rather that the action of a particular odorant is inherent in the olfactory receptor cell on which an odorant acts.     

Characterization of a phosphoinositide-mediated odor transduction pathway reveals plasma membrane localization of an inositol 1,4, 5-trisphosphate receptor in lobster olfactory receptor neurons

The role of phosphoinositide signaling in olfactory transduction is still being resolved. Compelling functional evidence for the transduction of odor signals via phosphoinositide pathways in olfactory transduction comes from invertebrate olfactory systems, in particular lobster olfactory receptor neurons. We now provide molecular evidence for two components of the phosphoinositide signaling pathway in lobster olfactory receptor neurons, a G protein alpha subunit of the G(q) family and an inositol 1,4, 5-trisphosphate-gated channel or an inositol 1,4,5-trisphosphate (IP(3)) receptor. Both proteins localize to the site of olfactory transduction, the outer dendrite of the olfactory receptor neurons. Furthermore, the IP(3) receptor localizes to membranes in the ciliary transduction compartment of these cells at both the light microscopic and electron microscopic levels. Given the absence of intracellular organelles in the sub-micron diameter olfactory cilia, this finding indicates that the IP(3) receptor is associated with the plasma membrane and provides the first definitive evidence for plasma membrane localization of an IP(3)R in neurons. The association of the IP(3) receptor with the plasma membrane may be a novel mechanism for regulating intracellular cations in restricted cellular compartments of neurons.     

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase Cδ1 (PLCδ1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P₂/IP₃ and PI(3,4,5)P₃, the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2 s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.     

Cyclic nucleotide-gated channel activation is not required for activity-dependent labeling of zebrafish olfactory receptor neurons by amino acids

The olfactory epithelium of fish is heterogeneous both with respect to the types of receptor cells (ORNs) present and the families of odorant receptors expressed in these cells. As a consequence of this diversity, the transduction cascade(s) activated by odorants has yet to be unambiguously established. In the current study, electrophysiological and activity-dependent labeling techniques were used to assess the role of the cyclic nucleotide-gated channel in zebrafish olfactory transduction. Both amino acid and bile salt odorants elicited robust electrophysiological responses, however, activity-dependent labeling of ORNs could be stimulated only by the amino acid odorants. An adenylate cyclase (AC) activator (forskolin) and a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) also elicited robust electrophysiological responses; generally larger than the responses elicited by either the amino acid or bile salt odorants. However, neither forskolin alone or a mixture of forskolin and IBMX stimulated activity-dependent labeling. Bathing the olfactory epithelium with forskolin, which presumably increased the intracellular concentration of cAMP, reduced the responses to bile salt odorants to a significantly greater extent than amino acid odorants. Collectively, these findings suggest that the transduction of amino acid input does not rely primarily on cyclic nucleotide-gated (CNG) channel activation and that CNG channel activation may be required for the transduction of bile salt input. Copyright Copyright 1999 S. Karger AG, Basel     

Negative regulation and gain control of sensory neurons by the C. elegans calcineurin TAX-6

Animals sense and adapt to variable environments by regulating appropriate sensory signal transduction pathways. Here, we show that calcineurin plays a key role in regulating the gain of sensory neuron responsiveness across multiple modalities. C. elegans animals bearing a loss-of-function mutation in TAX-6, a calcineurin A subunit, exhibit pleiotropic abnormalities, including many aberrant sensory behaviors. The tax-6 mutant defect in thermosensation is consistent with hyperactivation of the AFD thermosensory neurons. Conversely, constitutive activation of TAX-6 causes a behavioral phenotype consistent with inactivation of AFD neurons. In olfactory neurons, the impaired olfactory response of tax-6 mutants to an AWC-sensed odorant is caused by hyperadaptation, which is suppressible by a mutation causing defective olfactory adaptation. Taken together, our results suggest that stimulus-evoked calcium entry activates calcineurin, which in turn negatively regulates multiple aspects of sensory signaling.     

Modeling of molecular and cellular mechanisms involved in Ca2+ signal encoding in airway myocytes

In airway myocytes signal transduction via cytosolic calcium plays an important role. In relation with experimental results we review models of basic molecular and cellular mechanisms involved in the signal transduction from the myocyte stimulation to the activation of the contractile apparatus. We concentrate on mechanisms for encoding of input signals into Ca²⁺ signals and the mechanisms for their decoding. The mechanisms are arranged into a general scheme of cellular signaling, the so-called bow-tie architecture of signaling, in which calcium plays the role of a common media for cellular signals and links the encoding and decoding part. The encoding of calcium signals in airway myocytes is better known and is presented in more detail. In particular, we focus on three recent models taking into account the intracellular calcium handling and ion fluxes through the plasma membrane. The model of membrane conductances was originally proposed for predicting membrane depolarization and voltage-dependent Ca²⁺ influx triggered by initial cytosolic Ca²⁺ increase as observed on cholinergic stimulation. Cellular models of intracellular Ca²⁺ handling were developed to investigate the role of a mixed population of InsP₃ receptor isoforms and the cellular environment in the occurrence of Ca²⁺ oscillations, and the respective role of the sarcoplasmic reticulum, mitochondria, and cytosolic Ca²⁺-binding proteins in cytosolic Ca²⁺ clearance. Modeling the mechanisms responsible for the decoding of calcium signals is developed in a lesser extent; however, the most recent theoretical studies are briefly presented in relation with the known experimental results.     

昆虫气味受体研究进展

嗅觉在昆虫的多种行为中发挥关键作用。气味分子与嗅觉神经元树突上气味受体的结合,参与了昆虫嗅觉识别的初始过程。昆虫的嗅觉神经元表达两类气味受体： 一是传统气味受体,该类受体同源性较低,在少部分嗅觉神经元中表达; 二是Or83b家族受体,该类受体不感受气味,在不同昆虫间较为保守且在大多数嗅觉神经元中表达。目前,对于单个传统气味受体的气味分子配体特异性所知甚少; 对于Or83b家族受体,一般认为其可能具有将传统气味受体运送至嗅觉神经元树突膜上的功能。此外,有一些实验证据不支持昆虫气味受体为G蛋白偶联受体的观点。