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.     

Spatiotemporal Alterations in Primary Odorant Representations in Olfactory Marker Protein Knockout Mice

Olfactory marker protein (OMP) is highly and selectively expressed in primary olfactory sensory neurons (OSNs) across species, but its physiological function remains unclear. Previous studies in the olfactory epithelium suggest that it accelerates the neural response to odorants and may modulate the odorant-selectivity of OSNs. Here we used a line of gene-targeted mice that express the fluorescent exocytosis indicator synaptopHluorin in place of OMP to compare spatiotemporal patterns of odorant-evoked neurotransmitter release from OSNs in adult mice that were heterozygous for OMP or OMP-null. We found that these patterns, which constitute the primary neural representation of each odorant, developed more slowly during the odorant presentation in OMP knockout mice but eventually reached the same magnitude as in heterozygous mice. In the olfactory bulb, each glomerulus receives synaptic input from a subpopulation of OSNs that all express the same odor receptor and thus typically respond to a specific subset of odorants. We observed that in OMP knockout mice, OSNs innervating a given glomerulus typically responded to a broader range of odorants than in OMP heterozygous mice and thus each odorant evoked synaptic input to a larger number of glomeruli. In an olfactory habituation task, OMP knockout mice behaved differently than wild-type mice, exhibiting a delay in their onset to investigate an odor stimulus during its first presentation and less habituation to that stimulus over repeated presentations. These results suggest that the actions of OMP in olfactory transduction carry through to the primary sensory representations of olfactory stimuli in adult mice in vivo.     

Response patterns of single neurons in the tortoise olfactory epithelium and olfactory bulb

The responses to odor stimulation of 40 single units in the olfactory mucosa and of 18 units in the olfactory bulb of the tortoise (Gopherus polyphemus) were recorded with indium-filled, Pt-black-tipped microelectrodes. The test battery consisted of 27 odorants which were proved effective by recording from small bundles of olfactory nerve. Two concentrations of each odorant were employed. These values were adjusted for response magnitudes equal to those for amyl acetate at –2.5 and –3.5 log concentration in olfactory twig recording. Varying concentrations were generated by an injection-type olfactometer. The mucosal responses were exclusively facilitory with a peak frequency of 16 impulses/sec. 19 mucosal units responded to at least one odorant and each unit was sensitive to a limited number of odorants (1–15). The sensitivity pattern of each unit was highly individual, with no clear-cut types, either chemical or qualitative, emerging. Of the 18 olfactory bulb units sampled, all responded to at least one odorant. The maximum frequency observed during a response was 39 impulses/sec. The bulbar neurons can be classified into two types. There are neurons that respond exclusively with facilitation and others that respond with facilitation to some odorants and with inhibition to others. Qualitatively or chemically similar odorants did not generate similar patterns across bulbar units.     

Searching for the Ligands of Odorant Receptors

Through the sense of smell mammals can detect and discriminate between a large variety of odorants present in the surrounding environment. Odorants bind to a large repertoire of odorant receptors located in the cilia of olfactory sensory neurons of the nose. Each olfactory neuron expresses one single type of odorant receptor, and neurons expressing the same type of receptor project their axons to one or a few glomeruli in the olfactory bulb, creating a map of odorant receptor inputs. The information is then passed on to other regions of the brain, leading to odorant perception. To understand how the olfactory system discriminates between odorants, it is necessary to determine the odorant specificities of individual odorant receptors. These studies are complicated by the extremely large size of the odorant receptor family and by the poor functional expression of these receptors in heterologous cells. This article provides an overview of the methods that are currently being used to investigate odorant receptor–ligand interactions.     

A network of stimulatory and inhibitory Galpha-subunits regulates olfaction in Caenorhabditis elegans

The two pairs of sensory neurons of C. elegans, AWA and AWC, that mediate odorant attraction, express six Galpha-subunits, suggesting that olfaction is regulated by a complex signaling network. Here, we describe the cellular localization and functions of the six olfactory Galpha-subunits: GPA-2, GPA-3, GPA-5, GPA-6, GPA-13, and ODR-3. All except GPA-6 localize to sensory cilia, suggesting a direct role in sensory transduction. GPA-2, GPA-3, GPA-5, and GPA-6 are also present in cell bodies and axons and GPA-5 specifically localizes to synaptic sites. Analysis of animals with single- to sixfold loss-of-function mutations shows that olfaction involves a balance between multiple stimulatory and inhibitory signals. ODR-3 constitutes the main stimulatory signal and is sufficient for the detection of odorants. GPA-3 forms a second stimulatory signal in the AWA and AWC neurons, also sufficient for odorant detection. In AWA, signaling is suppressed by GPA-5. In AWC, GPA-2 and GPA-13 negatively and positively regulate signaling, respectively. Finally, we show that only ODR-3 plays a role in cilia morphogenesis. Defects in this process are, however, independent of olfactory behavior. Our findings reveal the existence of a complex signaling network that controls odorant detection by C. elegans.     

Responses to putative second messengers and odorants in water nose olfactory neurons of Xenopus laevis

Using the whole-cell mode of the patch-clamp technique, we attempted to record inward currents in response to cAMP, inositol 1,4, 5-trisphosphate (IP(3)) and odorants from sensory neurons in the olfactory epithelium of the Xenopus laevis lateral diverticulum (water nose). Dialysis of 100 microM of IP(3) induced inward currents, while dialysis of 1 mM of cAMP into olfactory neurons did not induce any response under the voltage-clamp conditions. Changes in membrane conductance were examined by applying ramp pulses. The slope of the current-voltage (I-V) curve during the IP(3)-induced response was steeper than that after the response, indicating that IP(3) increased the membrane conductance. The water nose olfactory neurons have been shown to respond to both amino acids and volatile odorants. The slopes of I-V curves during responses to amino acids and a volatile odorant, lilial, were similar to those before the responses, suggesting that the total membrane conductance was not changed during responses to amino acids and the volatile odorant.     

Odorant receptor expression in the mouse cerebral cortex

Mammalian odorant receptors have been known to be involved not only in odorant detection but also in neuronal development of olfactory sensory neurons. We have examined a possibility of odorant receptor expression in nonolfactory neurons in the mouse. Mouse odorant receptors (M71, C6, and OR3), two of which were already shown to be functionally activated by odorants in heterologous systems, were detected by polymerase chain reactions (PCRs) from the cerebral cortex but not from other brain tissues. Degenerate PCR further suggested that other odorant receptors were also expressed in the mouse cerebral cortex. One of these receptors showed high sequence-match with a putative chick odorant receptor OR7 transiently expressed in the notochord during development. In situ hybridization detected signals for M71 and C6 receptors in the layer II cortical pyramidal neurons located in the occipital pole. In the M71-IRES-tauLacZ mouse, in which M71 expression was genetically marked with tauLacZ, X-gal staining signals were mostly localized in the layer II neurons in the occipital pole, being consistent with the in situ hybridization result. Fluorescent immunohistochemistry using anti-beta-galactosidase antibody further detected the tauLacZ signals in the same cells. X-gal staining began at P3, peaked at P8, and continued to adults, although signals gradually decreased. These data showed that at least a few odorant receptors are expressed not only in olfactory sensory neurons but also in pyramidal neurons in the cerebral cortex, possibly playing an important role either in chemical detection of exogenous or endogenous ligands or in a developmental process such as axon guidance and target recognition.     

Molecular determinants of odorant receptor function in insects

The olfactory system of Drosophila melanogaster provides a powerful model to study molecular and cellular mechanisms underlying function of a sensory system. In the 1970s Siddiqi and colleagues pioneered the application of genetics to olfactory research and isolated several mutant Drosophila with odorant-specific defects in olfactory behaviour, suggesting that odorants are detected differentially by the olfactory system. Since then basic principles of olfactory system function and development have emerged using Drosophila as a model. Nearly four decades later we can add computational methods to further our understanding of how specific odorants are detected by receptors. Using a comparative approach we identify two categories of short amino acid sequence motifs: ones that are conserved family-wide predominantly in the C-terminal half of most receptors, and ones that are present in receptors that detect a specific odorant, 4-methylphenol, found predominantly in the N-terminal half. The odorant-specific sequence motifs are predictors of phenol detection in Anopheles gambiae and other insects, suggesting they are likely to participate in odorant binding. Conversely, the family-wide motifs are expected to participate in shared functions across all receptors and a mutation in the most conserved motif leads to a reduction in odor response. These findings lay a foundation for investigating functional domains within odorant receptors that can lead to a molecular understanding of odor detection.     

A computational study of odorant transport and deposition in the canine nasal cavity: implications for olfaction

Olfaction begins when an animal draws odorant-laden air into its nasal cavity by sniffing, thus transporting odorant molecules from the external environment to olfactory receptor neurons (ORNs) in the sensory region of the nose. In the dog and other macrosmatic mammals, ORNs are relegated to a recess in the rear of the nasal cavity that is comprised of a labyrinth of scroll-like airways. Evidence from recent studies suggests that nasal airflow patterns enhance olfactory sensitivity by efficiently delivering odorant molecules to the olfactory recess. Here, we simulate odorant transport and deposition during steady inspiration in an anatomically correct reconstructed model of the canine nasal cavity. Our simulations show that highly soluble odorants are deposited in the front of the olfactory recess along the dorsal meatus and nasal septum, whereas moderately soluble and insoluble odorants are more uniformly deposited throughout the entire olfactory recess. These results demonstrate that odorant deposition patterns correspond with the anatomical organization of ORNs in the olfactory recess. Specifically, ORNs that are sensitive to a particular class of odorants are located in regions where that class of odorants is deposited. The correlation of odorant deposition patterns with the anatomical organization of ORNs may partially explain macrosmia in the dog and other keen-scented species.     

Odorant Metabolism Catalyzed by Olfactory Mucosal Enzymes Influences Peripheral Olfactory Responses in Rats

A large set of xenobiotic-metabolizing enzymes (XMEs), such as the cytochrome P450 monooxygenases (CYPs), esterases and transferases, are highly expressed in mammalian olfactory mucosa (OM). These enzymes are known to catalyze the biotransformation of exogenous compounds to facilitate elimination. However, the functions of these enzymes in the olfactory epithelium are not clearly understood. In addition to protecting against inhaled toxic compounds, these enzymes could also metabolize odorant molecules, and thus modify their stimulating properties or inactivate them. In the present study, we investigated the in vitro biotransformation of odorant molecules in the rat OM and assessed the impact of this metabolism on peripheral olfactory responses. Rat OM was found to efficiently metabolize quinoline, coumarin and isoamyl acetate. Quinoline and coumarin are metabolized by CYPs whereas isoamyl acetate is hydrolyzed by carboxylesterases. Electro-olfactogram (EOG) recordings revealed that the hydroxylated metabolites derived from these odorants elicited lower olfactory response amplitudes than the parent molecules. We also observed that glucurono-conjugated derivatives induced no olfactory signal. Furthermore, we demonstrated that the local application of a CYP inhibitor on rat olfactory epithelium increased EOG responses elicited by quinoline and coumarin. Similarly, the application of a carboxylesterase inhibitor increased the EOG response elicited by isoamyl acetate. This increase in EOG amplitude provoked by XME inhibitors is likely due to enhanced olfactory sensory neuron activation in response to odorant accumulation. Taken together, these findings strongly suggest that biotransformation of odorant molecules by enzymes localized to the olfactory mucosa may change the odorant’s stimulating properties and may facilitate the clearance of odorants to avoid receptor saturation.     

Roles of odorant receptors in projecting axons in the mouse olfactory system

In the mouse olfactory epithelium, there are about ten million olfactory sensory neurons, each expressing a single type of odorant receptor out of approximately 1000. Olfactory sensory neurons expressing the same odorant receptor converge their axons to a specific set of glomeruli on the olfactory bulb. How odorant receptors play an instructive role in the projection of axons to the olfactory bulb has been one of the major issues of developmental neurobiology. Recent studies revealed previously overlooked roles of odorant receptor-derived cAMP signals in the axonal projection of olfactory sensory neurons; the levels of cAMP and neuronal activity appear to determine the expression levels of axon guidance/sorting molecules and thereby direct the axonal projection of olfactory sensory neurons. These findings provide new insights as to how peripheral inputs instruct neuronal circuit formation in the mammalian brain.     

Spatial representation of odorant valence in an insect brain

Brains have to decide whether and how to respond to detected stimuli based on complex sensory input. The vinegar fly Drosophila melanogaster evaluates food sources based on olfactory cues. Here, we performed a behavioral screen using the vinegar fly and established the innate valence of 110 odorants. Our analysis of neuronal activation patterns evoked by attractive and aversive odorants suggests that even though the identity of odorants is coded by the set of activated receptors, the main representation of odorant valence is formed at the output level of the antennal lobe. The topographic clustering within the antennal lobe of valence-specific output neurons resembles a corresponding domain in the olfactory bulb of mice. The basal anatomical structure of the olfactory circuit between insects and vertebrates is known to be similar; our study suggests that the representation of odorant valence is as well.     

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors^1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.     

Zonal organization of the mammalian main and accessory olfactory systems

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.     

Advantage of the Highly Restricted Odorant Receptor Expression Pattern in Chemosensory Neurons of Drosophila

A fundamental molecular feature of olfactory systems is that individual neurons express only one receptor from a large odorant receptor gene family. While numerous theories have been proposed, the functional significance and evolutionary advantage of generating a sophisticated one-receptor-per neuron expression pattern is not well understood. Using the genetically tractable Drosophila melanogaster as a model, we demonstrate that the breakdown of this highly restricted expression pattern of an odorant receptor in neurons leads to a deficit in the ability to exploit new food sources. We show that animals with ectopic co-expression of odorant receptors also have a competitive disadvantage in a complex environment with limiting food sources. At the level of the olfactory system, we find changes in both the behavioral and electrophysiological responses to odorants that are detected by endogenous receptors when an olfactory receptor is broadly misexpressed in chemosensory neurons. Taken together these results indicate that restrictive expression patterns and segregation of odorant receptors to individual neuron classes are important for sensitive odor-detection and appropriate olfactory behaviors.     

Developmental regulation of olfactory circuit formation in mice

In mammals, odorants induce various behavioral responses that are critical to the survival of the individual and species. Binding signals of odorants to odorant receptors (ORs) expressed in the olfactory epithelia are converted to an odor map, a pattern of activated glomeruli, in the olfactory bulb (OB). This topographic map is used to identify odorants for memory-based learned decisions. In the embryo, a coarse olfactory map is generated in the OB by a combination of dorsal-ventral and anterior-posterior targeting of olfactory sensory neurons (OSNs), using specific sets of axon-guidance molecules. During the process of OSN projection, odor signals are sorted into distinct odor qualities in separate functional domains in the OB. Odor information is then conveyed by the projection neurons, mitral/tufted cells, to various regions in the olfactory cortex, particularly to the amygdala for innate olfactory decisions. Although the basic architecture of hard-wired circuits is generated by a genetic program, innate olfactory responses are modified by neonatal odor experience in an activity-dependent manner. Stimulus-driven OR activity promotes post-synaptic events and dendrite selection in the responding glomeruli making them larger. As a result, enhanced odor inputs in neonates establish imprinted olfactory memory that induces attractive responses in adults, even when the odor quality is innately aversive. In this paper, I will provide an overview of the recent progress made in the olfactory circuit formation in mice.     

A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.

The mammalian olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants presumably results from the association of odorous ligands with specific receptors on olfactory sensory neurons. To address the problem of olfactory perception at a molecular level, we have cloned and characterized 18 different members of an extremely large multigene family that encodes seven transmembrane domain proteins whose expression is restricted to the olfactory epithelium. The members of this novel gene family are likely to encode a diverse family of odorant receptors.     

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust,Locusta migratoria

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.     

Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe

A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant-receptor gene converge onto the same glomeruli in the brain [1-7]. Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants [8-11]. OSNs also exhibit odor-evoked responses with complex temporal dynamics [11], but the contribution of this activity to behavioral odor discrimination has received little attention [12]. Here, we investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. We show that Drosophila can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies encode one odorant from a mixture and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants by using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration, indicating that they encode odorant identity. Therefore, flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination.