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.     

Effects of in utero odorant exposure on neuroanatomical development of the olfactory bulb and odour preferences

Human babies and other young mammals prefer food odours and flavours of their mother's diet during pregnancy as well as their mother's individually distinctive odour. Newborn mice also prefer the individual odours of more closely related--even unfamiliar--lactating females. If exposure to in utero odorants-which include metabolites from the mother's diet and the foetus's genetically determined individual odour-helps shape the neuroanatomical development of the olfactory bulb, this could influence the perception of such biologically important odours that are preferred after birth. We exposed gene-targeted mice during gestation and nursing to odorants that activate GFP-tagged olfactory receptors (ORs) and then measured the effects on the size of tagged glomeruli in the olfactory bulb where axons from olfactory sensory neurons (OSNs) coalesce by OR type. We found significantly larger tagged glomeruli in mice exposed to these activating odorants in amniotic fluid, and later in mother's milk, as well as significant preferences for the activating odour. Larger glomeruli comprising OSNs that respond to consistently encountered odorants should enhance detection and discrimination of these subsequently preferred odours, which in nature would facilitate selection of palatable foods and kin recognition, through similarities in individual odours of relatives.     

Asymmetrical generalisation between pheromonal and floral odours in appetitive olfactory conditioning of the honey bee (Apis mellifera L.)

The capacity to generalise between similar but not identical olfactory stimuli is crucial for honey bees, allowing them to find rewarding food sources with varying volatile emissions. We studied bees' generalisation behaviour with odours having different biological values: typical floral odours or alarm compounds. Bees' behavioural and peripheral electrophysiological responses were investigated using a combined proboscis extension response conditioning-electroantennogram assay. Bees were conditioned to pure linalool (floral) or to pure isoamyl acetate (alarm) and were tested with different concentrations of both compounds. Electrophysiological responses were not influenced by conditioning, suggesting that the learning of individual compounds does not rely on modulations of peripheral sensitivity. Behaviourally, generalisation responses of bees conditioned to the alarm compound were much higher than those of bees conditioned to the floral odour. We further demonstrated such asymmetrical generalisation between alarm and floral odours by using differential conditioning procedures. Conditioning to alarm compounds (isoamyl acetate or 2-heptanone) consistently induced more generalisation than conditioning to floral compounds (linalool or phenylacetaldehyde). Interestingly, generalisation between the two alarm compounds, which are otherwise chemically different, was extremely high. These results are discussed in relation to the neural representation of compounds with different biological significance for bees.     

Learning-induced modulation of oscillatory activities in the mammalian olfactory system: The role of the centrifugal fibres

In the mammalian olfactory system, oscillations related to odour representation have been described in field potential activities. Previous results showed that in olfactory bulb (OB) of awake rats engaged in an olfactory learning, odour presentation produced a decrease of oscillations in gamma frequency range (60-90 Hz) associated with a power increase in beta frequency range (15-40 Hz). This response pattern was strongly amplified in trained animals. The aim of this work was twofold: whether learning also induces similar changes in OB target structures and whether such OB response depends on its centrifugal inputs. Local field potentials (LFPs) were recorded through chronically implanted electrodes in the OB, piriform and enthorhinal cortices of freely moving rats performing an olfactory discrimination. Oscillatory activities characteristics (amplitude, frequency and time-course) were extracted in beta and gamma range by a wavelet analysis. First, we found that odour induced beta oscillatory activity was present not only in the OB, but also in the other olfactory structures. In each recording site, characteristics of the beta oscillatory responses were dependent of odour, structure and learning level. Unilateral section of the olfactory peduncle was made before training, and LFPs were symmetrically recorded in the two bulbs all along the acquisition of the learning task. Data showed that deprivation of centrifugal feedback led to an increase of spontaneous gamma activity. Moreover, under this condition olfactory learning was no longer associated with the typical large beta band. As a whole, learning modulation of the beta oscillatory response in olfactory structures may reflect activity of a distributed functional network involved in odour representation.     

OLFACTORY BULB RESPONSIVENESS TO FOOD ODOUR DURING STOMACH DISTENSION IN THE RAT

In the rat's olfactory bulb, the mitral cell multiunit activityin response to food odour changes with the internal states offood deprivation or satiety. It is demonstrated that stomachdistension should be one of the factors acting on the centrifugalcontrol of the olfactory input. An inflatable balloon was chronicallyinserted into the stomach of male rats trained to eat a singledaily meal. The mitral cell responses to the usual food odourand to the control odour of isoamyl acetate were recorded inunanaesthetized fasted animals for 3 successive states of thestomach: empty, then inflated by introducing water into theballoon, and empty again. Stomach inflation produced a rapid,long lasting and significant decrease of the bulb responsivenessto food odour without changing the responsiveness to controlodour. Stomach distension thus simulates the previously noticedeffects of satiety on the selective modulation of the olfactoryinput.     

Electrophysiological responses of olfactory bulb neurons to odour stimuli in the frog. A comparison with receptor cells

Extracellular recordings were performed from olfactory bulbneurons in the frog. The odour stimuli were the same as thosepreviously used for studying the receptor cells in the sameanimal species and were delivered at similar concentrations(Revial et al., 1982). The general properties of the neuronresponses are presented and discussed with reference to homologousproperties of olfactory receptor cells. The response rates elicitedby different stimuli from the bulbar neurons were found to behighly correlated with those elicited from receptor cells. Theindividual cell selectivity was better in the bulb than in theolfactory epithelium. The olfactory bulb neurons seemed to improvethe discrimination between stimuli (enantiomers) poorly distinguishedby the receptor cells. Reducing odor concentration caused therate of suppressive response to decrease faster than that ofexcitatory ones, suggesting that the manifestations of inhibitoryprocesses in some neurons requires a high level of excitationin others.     

Ontogenetic development of neural responses in the olfactory bulb of laboratory mice

Summary In laboratory mice (strain NMRI) the ontogenetic development of single unit activity in the olfactory bulb was investigated. From postnatal day 10 on, spontaneously active neurons were recorded with glass-microelectrodes, and their responses to olfactory stimuli were tested (butyric acid, geraniol, grass- and nest-odour).From day 10 to 13 only very few neurons were recordable (and most of these elements were too weak and were lost before being stimulated). At day 14 the number of recordable neurons increased rapidly, and by day 15 spontaneously active neurons reached adult level in terms of incidence and electric properties.There were 3 types of neurons: 1. respiration synchronous elements; 2. bursting neurons not correlated with respiration; 3. continuously, but randomly, firing elements (about 60% of all neurons). Reactions to odour stimuli (excitation, ca. 50%; inhibition, ca. 34%; complex reactions, ca. 12%; change in activity pattern, ca. 4%) occurred as soon as the cells were stable enough for testing. The reaction patterns showed no age specific differences; the duration of the responses varied from 100 ms to 100 s.In younger animals (P11–P14) the percentage of responses was slightly smaller (47%) than in the older ones (P30–P50; 64% response to olfactory stimulation). For some of the odours tested the proportion of responding cells differed depending on age (for instance grass odour evoked a response in 40% of the cells in young ones, but in 65% in adults).Abbreviations AP action potential - In interneuron - MTc mitral or tufted cell - P10 postnatal day 10     

A chemical-modification approach to the olfactory code. Studies with a thiol-specific reagent

The effects of thiol-specific reagents on the amplitude of the electro-olfactogram (E.O.G.) responses elicited from frog olfactory mucosa by pulses of odorant vapours was studied. The impermeant thiol-specific reagent mersalyl [(3-{[2-(carboxymethoxy)-benzoyl]amino}-2-methoxypropyl)hydroxymercury monosodium salt] brings about a rapid decrease in the E.O.G. signal obtained with the odorant pentyl acetate. The extent of the decrease is proportional to the concentration of the mersalyl applied and the effect of the reagent is partially but incompletely reversed by treatment of the labelled mucosa with dithiothreitol. The sites labelled by mersalyl can be protected by pretreating the mucosa with a dilute solution of the odorant pentyl acetate and leaving the solution in contact with the tissue after the addition of mersalyl. When the protecting odorant is washed out of the tissue, the original E.O.G. amplitude is regained. Pentyl acetate applied to the mucosa protected the E.O.G. response to vapour pulses of the following odorants from the effects of mersalyl: n-butyric acid, n-butyl acetate, phenylacetaldehyde and cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane). The pentyl acetate applied to the mucosa failed to protect the E.O.G. response to vapour pulses of the following odorants from the effects of mersalyl: butan-1-ol, benzyl acetate, nitrobenzene, beta-ionone and linalyl acetate. The significance of the differential protection effects for the odour-quality-coding mechanism in the olfactory primary neurons is discussed. It is suggested that the olfactory code at this level of the olfactory system may be elucidated by chemical-modification methods.     

Second messenger-controlled membrane conductance in locust (Locusta migratoria) olfactory neurons

The membrane conductance of olfactory neurons of Locusta migratoria was examined using the whole-cell configuration of the patch-clamp technique. Intracellular application of the second messenger inositol 1,4,5 trisphosphate (IP(3)) via a dual pipette technique elicited a clear increase in the membrane conductance. The IP(3)-induced conductance increased due to a rise in the extracellular concentration of calcium from 100&mgr;M to 4mM. Micromolar concentrations of ruthenium red partially blocked the IP(3)-induced increase in membrane conductance. Stimulating olfactory neurons with odour (hexenoic acid) resulted in an increase in the membrane conductance partially similar to that mediated IP(3). These findings suggest that stimulation with appropriate odours as well as intracellular application of IP(3) activate the same calcium-permeable ion channels in the plasma membrane of insect olfactory neurons.     

Regeneration of olfactory flagella and restoration of the electroolfactogram following application of triton X-100 to the olfactory mucosa of frogs]

A short-tern (1-1.5 min.) irrigation of the olfactory mucose of the frog Rana temporaria with 0.1-0.15% Triton X-100 in Ringer's solution led to the destroying of olfactory flagella but did not damage the olfactory knob and its flagellar basal bodies. Simultaneously, the generator potential of the olfactory cells-elecroolfactogram (EOG)-disappears. The olfactory cells deprived of fragella were able to produce these organelles. This process begins 2 or 3 hours following theflagellum removal, proceeds in some stages and completes within 2 or 3 days. During the flagellum regeneration the ability of olfactory cells to generate EOG is seen to resotre. The data obtained confirm the presence of receptive sites on flagellar surface.     

Electrophysiological characterization of olfactory cell types in the antennae and palps of the housefly

A set of odours was presented to the housefly Musca domestica and the electrophysiological responses of single olfactory receptor cells in the antennae and palps were recorded. The olfactory cells in the antennae of the housefly showed a large variability of response profiles, but multidimensional cluster analysis suggested a moderate clustering in olfactory response types. Receptor cells with similar or with different odour response profiles can reside in one and the same sensillum. No fixed spatial distribution of olfactory response types over the antennal of palpal surface was found. The odours of 1-octen-3-ol, amyl acetate, 3-methylphenol, 2-pentanone and R(+)limonene elicited the largest responses in antennal cells. Most odours elicited responses in cells of only a few of the clusters, but 1-octen-3-ol was detected by cells of almost all clusters of the antenna. Surprisingly, rather low responses were found to acetic acid, skatole, indole and muscalure, odours that are known to attract flies. Response profiles of palpal cells differed considerably from those of antennal cells. Palpal cells mostly responded to 3-methylphenol and 2-pentanone. In the palps, the clusters of cells responding to 3-methylphenol and 2-pentanone are clearly separated from the other olfactory cells.     

Odours stimulate neuronal activity in the dorsolateral area of the hippocampal formation during path integration

The dorsolateral area of the hippocampal formation of birds is commonly assumed to play a central role in processing information needed for geographical positioning and homing. Previous work has interpreted odour-induced activity in this region as evidence for an ‘olfactory map’. Here, we show, using c-Fos expression as a marker, that neuronal activation in the dorsolateral area of the hippocampal formation of pigeons is primarily a response to odour novelty, not to the spatial distribution of odour sources that would be necessary for an olfactory map. Pigeons exposed to odours had significantly more neurons activated in this area of the brain than pigeons exposed to filtered air with odours removed. This increased activity was observed only in response to unfamiliar odours. No change in activity was observed when pigeons were exposed to home odours. These findings are consistent with non-home odours activating non-olfactory components of the pigeon''s navigation system. The pattern of neuronal activation in the triangular and dorsomedial areas of the hippocampal formation was, by contrast, consistent with the possibility that odours play a role in providing spatial information.     

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.     

Adult frogs are sensitive to the predation risks of olfactory communication

Olfaction is a common sensory mode of communication in much of the Vertebrata, although its use by adult frogs remains poorly studied. Being part of an open signalling system, odour cues can be exploited by 'eavesdropping' predators that hunt by smell, making association with odour a high-risk behaviour for prey. Here, we show that adult great barred frogs (Mixophes fasciolatus) are highly attracted to odour cues of conspecifics and those of sympatric striped marsh frogs (Limnodynastes peronii). This attraction decreased significantly with the addition of odours of a scent-hunting predator, the red-bellied black snake (Pseudechis porphyriacus), indicating that frogs perceived predation risks from associating with frog odours. Male frogs, however, maintained some attraction to unfamiliar conspecific scents even with predator odours present, suggesting that they perceived benefits of odour communication despite the risk. Our results indicate that adult frogs can identify species and individuals from their odours and assess the associated predation risk, revealing a complexity in olfactory communication previously unknown in adult anurans.     

猫嗅球外丛层和僧帽细胞层年龄相关形态学变化

分别用Nissl法及免疫组织化学ABC法标记青、老年猫嗅球中嗅觉二级神经元和外丛层胶质细胞,显微镜下观察其分布并计数,对嗅觉二级神经元胞体直径和外丛层厚度进行测量,比较其年龄相关性变化,研究神经元与胶质细胞之间的关系,探讨老年性嗅觉功能衰退的相关神经机理。结果显示,老年猫嗅觉二级神经元胞体直径和分布密度均有不同程度的显著性下降(P<0.05);外丛层厚度变化不明显(P>0.05);外丛层胶质细胞特别是星形胶质细胞显著性增生(P<0.05)。表明在衰老过程中嗅觉二级神经元有丢失,并呈现功能下降,可能是老年性嗅觉功能衰退的原因之一。同时外丛层胶质细胞增生以进一步保护神经元,延缓其衰老。     

Electrophysiological demonstration of independent olfactory receptor types and associated neuronal responses in the trout olfactory bulb

The present study attempts to highlight the principles by which peripheral olfactory information of across- and within-class odorant signals is transformed into bulbar neuron responses. For this purpose, we performed electro-olfactogram cross-adaptation and mixture experiments as well as single unit recording of olfactory bulb neurons using amino acid, bile acid and F-prostaglandin stimulants in brown and rainbow trout. The results show that amino acids, a bile acid and a F-prostaglandin activate independent receptor types. However, within the class of amino acids, different receptor types are only partially independent. Neurons responsive to bile acid and amino acids were segregated to the mid-dorsal and latero-posterior olfactory bulb, respectively. Of the 43 responsive olfactory bulb neurons studied in brown trout, 41 showed specificity for one odorant class. Olfactory bulb neurons gained responsiveness to new amino acids with increasing stimulant concentration. We conclude that different odorant classes activate specific neurons located in different regions of the trout olfactory bulb, and that information distinguishing related amino acids can be represented in a limited number of bulbar neurons with distinct response profiles under the conditions investigated.     

Functional regeneration of the olfactory bulb requires reconnection to the olfactory nerve in Xenopus larvae

Larvae of the South African clawed frog (Xenopus laevis) can regenerate the telencephalon, which consists of the olfactory bulb and the cerebrum, after it has been partially removed. Some authors have argued that the telencephalon, once removed, must be reconnected to the olfactory nerve in order to regenerate. However, considerable regeneration has been observed before reconnection. Therefore, we have conducted several experiments to learn whether or not reconnection is a prerequisite for regeneration. We found that the olfactory bulb did not regenerate without reconnection, while the cerebrum regenerated by itself. On the other hand, when the brain was reconnected by the olfactory nerve, both the cerebrum and the olfactory bulb regenerated. Morphological and histological investigation showed that the regenerated telencephalon was identical to the intact one in morphology, types and distributions of cells, and connections between neurons. Froglets with a regenerated telencephalon also recovered olfaction, the primary function of the frog telencephalon. These results suggest that the Xenopus larva requires reconnection of the regenerating brain to the olfactory nerve in order to regenerate the olfactory bulb, and thus the regenerated brain functions, in order to process olfactory information.     

Exposure to behaviourally relevant odour reveals differential characteristics in rat central olfactory pathways as studied through oscillatory activities

This study investigated how changes in nutritional motivation modulate odour-related oscillatory activities at several levels of the olfactory pathway in non-trained rats. Local field potential recordings were obtained in freely moving animals in the olfactory bulb (OB), anterior and posterior parts of the piriform cortex (APC and PPC respectively) and lateral entorhinal cortex (EC). Dynamic signal analysis detected changes in power during odour presentation for several frequency bands The results showed that in most cases odour presentation was associated with changes in a wide 15-90 Hz frequency band of activity in each olfactory structure. However, nutritional state modulated initial responses to food odour (FO) in the OB and EC selectively in the 15-30 Hz frequency band. Changes in nutritional state also modulated responses to repeated FO stimuli. Habituation was expressed differentially across structures with a clear dissociation between the two parts of the piriform cortex. Finally, systemic injections of scopolamine (0.125 mg/kg) selectively blocked expression of the nutritional modulation in the OB found in the beta band. These results suggest that internal state can differentially modulate odour processing among different olfactory areas and point to a cholinergic-sensitive beta band oscillation during presentation of a behaviourally meaningful odorant.     

Odorant-induced hyperpolarization and suppression of cAMP-activated current in newt olfactory receptor neurons

Although many studies have reported that odorants can elicit inhibitory responses as well as excitatory responses in vertebrate olfactory receptor neurons, the cellular mechanisms that underlie this inhibition are unclear. Here we examine the inhibitory effect of odorants on newt olfactory receptor neurons using whole cell patch clamp recording. At high concentrations, odorant stimulation decreased the membrane conductance and inhibited depolarization. Various odorants (anisole, isoamyl acetate, cineole, limonene and isovaleric acid) suppressed the depolarizing current in a dose-dependent manner. Furthermore, one odorant could suppress the depolarization caused by another odorant. The depolarization caused by isoamyl acetate was inhibited by anisole in cells that were excited by isoamyl acetate but not by anisole. Odorants were able to hyperpolarize cells that were depolarized by cAMP-induced conductance. Given that this inhibitory effect of odorants can affect excitation caused by other odorants, we suggest that it might play a role in coding odorants in olfactory receptor neurons.     

Axon Guidance Events in the Wiring of the Mammalian Olfactory System

The detection of odorant signals from the environment and the generation of appropriate behavioral outputs in response to these signals rely on the olfactory system. Olfactory sensory neurons (OSNs) of the olfactory epithelium are located in the nasal cavity and project axons that synapse onto dendrites of second-order neurons in the olfactory bulb (OB) that in turn relay the information gathered to higher order regions of the brain. The connections formed are remarkably accurate such that axons of OSNs expressing the same olfactory receptor innervate specific glomeruli within the complex three-dimensional structure that represents the OB. The molecular determinants that control this complex process are beginning to be identified. In this review, we discuss the role of various families of axon guidance cues and of recently characterized families of adhesion molecules in the formation of stereotypic connections in the olfactory system of mice. Cho and Prince contributed equally.