"Imposed" and "inherent" mucosal activity patterns. Their composite representation of olfactory stimuli

Both regional differences in mucosal sensitivity and a gas chromatography-like process along the mucosal sheet have been separately proposed in two sets of earlier studies to produce different odorant-dependent activity patterns across the olfactory mucosa. This investigation evaluated, in one study, whether and to what degree these two mechanisms contribute to the generation of these activity patterns. Summated multiunit discharges were simultaneously recorded from lateral (LN) and medial (MN) sites on the bullfrog's olfactory nerve to sample the mucosal activity occurring near the internal and external nares, respectively. Precisely controlled sniffs of four odorants (benzaldehyde, butanol, geraniol, and octane) were drawn through the frog's olfactory sac in both the forward (H1) and reverse (H2) hale directions. By combining the four resulting measurements, LNH1, LNH2, MNH1, and MNH2, in different mathematical expressions, indexes reflecting the relative effects of the chromatographic process, regional sensitivity, and hale direction could be calculated. Most importantly, the chromatographic process and the regional sensitivity differences both contributed significantly to the mucosal activity patterns. However, their relative roles varied markedly among the four odorants, ranging from complete dominance by either one to substantial contributions from each. In general, the more strongly an odorant was sorbed by the mucosa, the greater was the relative effect of the chromatographic process; the weaker the sorption, the greater the relative effect of regional sensitivity. Similarly, the greater an odorant's sorption, the greater was the effect of hale direction. Other stimulus variables (sniff volume, sniff duration, and the number of molecules within the sniff) had marked effects upon the overall size of the response. For strongly sorbed odorants, the effect of increasing volume was positive; for a weakly sorbed odorant, it was negative. The reverse may be true for duration. In contrast, the effect of increasing the number of molecules was uniformly positive for all four odorants. However, there was little evidence that these other stimulus variables had a major influence upon the effects of the chromatographic process and regional sensitivity differences in their generation of mucosal activity patterns.     

Factors influencing the differential sorption of odorant molecules across the olfactory mucosa

By use of a flow dilution olfactometer, tritium-labeled odorants were presented through the external naris to the bullfrog's intact olfactory sac. After stimulation the animal was frozen in liquid nitrogen. The dorsal surface and eminentia of the olfactory sac were then removed and sawed into sections perpendicular to the long axis of the mucosal surface. Each section was dissolved in a tissue solubilizer and counted in a liquid scintillation system. The amount of radioactivity in each section was used to estimate the number of odorant molecules it sorbed. For tritiated butanol there was a significant decrease in radioactivity from the section containing the external naris to that overhanging the internal naris. The steepness of the gradient was unaffected by a rather large range of stimulus flow rates, volumes, and partial pressures. Only when these parameters were pushed to extreme physical limits did this gradient change significantly. When the stimulus was presented through the internal rather than the external naris, the butanol gradient reversed its direction, decreasing from the internal to external. Unlike butanol, tritiated octane presented through the external naris was rather evenly distributed among the mucosal sections. That is, octane showed no distribution gradient across the mucosa. These results complement previous electrophysiological data that suggested a "chromatographic-like" differential sorption of odorant molecules across the mucosa.     

Evidence for a Chromatographic Model of Olfaction

The gradient of activity produced along the olfactory mucosa by odorant stimulation was measured by the ratio (the LB/MB ratio) of the summated neural discharges recorded from two branches of the olfactory nerve, a lateral branch (LB) supplying a mucosal region near the internal naris and a medial branch (MB) supplying a region near the external naris. Twenty-four frogs "sniffed" sixteen different odorants, each odorant at four concentrations and two flow rates. Increases in concentration and flow rate produced statistically reliable increases in the ratios; the magnitude of these increases was considerably smaller than the magnitude of the statistically significant changes that could be achieved by shifting the odorants themselves. Even the small change due to concentration depended upon the odorant presented. Thus, even at the highest physiologically possible concentrations and flow rates, the general level of the activity gradient along the mucosa appeared to be determined mainly by the particular odorant used. The relative retention time of each of these 16 different odorants was measured in a gas chromatograph fitted with a Carbowax 20M column. In general, the longer the odorant's retention time the smaller its LB/MB ratio. This suggests that the different mucosal gradients of activity are established for different odorants by a chromatographic process. The data further suggest that the mucosa behaves like a polar chromatographic column.     

Numerical modeling of odorant uptake in the rat nasal cavity

An anatomically accurate 3-dimensional numerical model of the right rat nasal cavity was developed and used to compute low, medium, and high flow rate inspiratory and expiratory mucosal odorant uptake (imposed patterning) for 3 odorants with different mucus solubilities. The computed surface mass flux distributions were compared with anatomic receptor gene expression zones identified in the literature. In general, simulations predicted that odorants that were highly soluble in mucus were absorbed dorsally and medially, corresponding roughly to receptors from one of the gene expression zones. Insoluble odorants tended to be absorbed more peripherally in the rat olfactory region corresponding to the other 2 zones. These findings also agreed in general with the electroolfactogram measurements and the voltage-sensitive dye measurements reported in the literature. This numerical approach is the first to predict detailed odorant flux information across the olfactory mucosa in the rat nasal cavity during inspiratory and expiratory flow and to relate it to anatomic olfactory receptor location, physiological function, and biochemical experiment. This numerical technique can allow us to separate the contributions of imposed and inherent patterning mechanisms on the rat olfactory mucosa.     

Numerical modeling of turbulent and laminar airflow and odorant transport during sniffing in the human and rat nose

Human sniffing behavior usually involves bouts of short, high flow rate inhalation (>300 ml/s through each nostril) with mostly turbulent airflow. This has often been characterized as a factor enabling higher amounts of odorant to deposit onto olfactory mucosa than for laminar airflow and thereby aid in olfactory detection. Using computational fluid dynamics human nasal cavity models, however, we found essentially no difference in predicted olfactory odorant flux (g/cm2 s) for turbulent versus laminar flow for total nasal flow rates between 300 and 1000 ml/s and for odorants of quite different mucosal solubility. This lack of difference was shown to be due to the much higher resistance to lateral odorant mass transport in the mucosal nasal airway wall than in the air phase. The simulation also revealed that the increase in airflow rate during sniffing can increase odorant uptake flux to the nasal/olfactory mucosa but lower the cumulative total uptake in the olfactory region when the inspired air/odorant volume was held fixed, which is consistent with the observation that sniff duration may be more important than sniff strength for optimizing olfactory detection. In contrast, in rats, sniffing involves high-frequency bouts of both inhalation and exhalation with laminar airflow. In rat nose odorant uptake simulations, it was observed that odorant deposition was highly dependent on solubility and correlated with the locations of different types of receptors.     

Experimental and numerical determination of odorant solubility in nasal and olfactory mucosa

Odorant deposition in the nasal and olfactory mucosas is dependent on a number of factors including local air/odorant flow distribution patterns, odorant mucosal solubility and odorant diffusive transport in the mucosa. Although many of these factors are difficult to measure, mucosal solubility in the bullfrog mucus has been experimentally determined for a few odorants. In the present study an experimental procedure was combined with computational fluid dynamic (CFD) techniques to further describe some of the factors that govern odorant mucosal deposition. The fraction of odorant absorbed by the nasal mucosa (eta) was experimentally determined for a number of odorants by measuring the concentration drop between odorant 'blown' into one nostril and that exiting the contralateral nostril while the subject performed a velopharyngeal closure. Odorant concentrations were measured with a photoionization detector. Odorants were delivered to the nostrils at flow rates of 3.33 and 10 l/min. The velopharyngeal closure nasal air/odorant flows were then simulated using CFD techniques in a 3-D anatomically accurate human nose modeland the mucosal odorant uptake was numerically calculated. The comparison between the numerical simulations and the experimental results lead to an estimation of the human mucosal odorant solubility and the mucosal effective diffusive transport resistance. The results of the study suggest that the increase in diffusive resistance of the mucosal layer over that of a thin layer of water seemed to be general and non-odorant-specific; however, the mucosa solubility was odorant specific and usually followed the trend that odorants with lower water solubility were more soluble in the mucosa than would be predicted from water solubility alone. The ability of this approach to model odorant movement in the nasal cavity was evaluated by comparison of the model output with known values of odorant mucosa solubility.     

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.     

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.     

The effect of concanavalin A on the rat electro-olfactogram at various odorant concentrations.

We have studied the effect of concanavalin A (Con A) on the rat electro-olfactogram response to several odorants. Each odorant was applied over a range of concentrations. For hydrophobic odorants whose response was affected by Con A, the diminution in response was maximal at odorant concentrations of about 1 microM in the olfactory mucus. The (odour) concentration-dependence of the change is compatible with the idea that Con A inactivates one or more types of olfactory receptor that normally bind odorants with dissociation constants of the order of 100 nM. With hydrophilic odorants we had to apply concentrations very much higher than this to elicit any response from the system. At these high concentrations we could observe Con A-induced diminutions in response.     

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.     

Physical Variables in the Olfactory Stimulation Process

Electrical recording from small twigs of nerve in a tortoise showed that olfactory, vomeronasal, and trigeminal receptors in the nose are responsive to various odorants. No one kind of receptor was most sensitive to all odorants. For controlled stimulation, odorant was caused to appear in a stream of gas already flowing through the nose. Of the parameters definable at the naris, temperature, relative humidity, and nature of inert gas had little effect on olfactory responses to amyl acetate, whereas odorant species, odorant concentration, and volume flow rate effectively determined the responses of all nasal chemoreceptors. An intrinsic variable of accessibility to the receptors, particularly olfactory, was demonstrated. Flow dependence of chemoreceptor responses is thought to reflect the necessity for delivery of odorant molecules to receptor sites. Since the olfactory receptors are relatively exposed, plateauing of the response with flow rate for slightly soluble odorants suggests an approach to concentration equilibrium in the overlying mucus with that in the air entering the naris. Accordingly, data for responses to amyl acetate were fitted with Beidler's (1954) taste equation for two kinds of sites being active. The requirement for finite aqueous solubility, if true, suggests substitution of aqueous solutions for gaseous solutions. A suitable medium was found and results conformed to expectations. Olfactory receptors were insensitive to variation of ionic strength, pH, and osmotic pressure.     

Effects of electrical stimulation of the human olfactory mucosa

Electrical stimulation of the human olfactory mucosa was performed by means of an electrode attached to a rhinoscope . Stimulation of the nasal mucosa did not evoke smell sensations, but suppressed smell sensations of presented odorants. When electrical stimulation followed the exposure to an odorant within a certain interval, the stimulus recalled the already faded sensation of the preceding odorant. Electrical stimulation without prior natural stimulation produced unpleasant sensations in 3 patients with a history of temporal lobe seizures and olfactory auras , but not in patients with primary, generalized or focal epilepsy.     

Sniffing and spatiotemporal coding in olfaction

The act of sniffing increases the air velocity and changes the duration of airflow in the nose. It is not yet clear how these changes interact with the intrinsic timing within the olfactory bulb, but this is a matter of current research activity. An action of sniffing in generating a high velocity that alters the sorption of odorants onto the lining of the nasal cavity is expected from the established work on odorant properties and sorption in the frog nose. Recent work indicates that the receptor properties in the olfactory epithelium and olfactory bulb are correlated with the receptor gene expression zones. The responses in both the epithelium and the olfactory bulb are predictable to a considerable extent by the hydrophobicity of odorants. Furthermore, receptor expression in both rodent and salamander nose interacts with the shapes of the nasal cavity to place the receptor sensitivity to odorants in optimal places according to the aerodynamic properties of the nose.     

A parametric study of the stimulation variables affecting the magnitude of the olfactory nerve response

The magnitude of olfactory responses can be related to three primary variables [number of odorant molecules (N), sniff volume (V), and sniff duration (T)] and three derived variables [concentration (C = N/V), flow rate (F = V/T), and delivery rate (D = N/T)]. To evaluate the effects of these interdependent variables upon the olfactory response, the summated multiunit discharges were recorded from the olfactory nerves of nine frogs in response to octane presented at two levels (in 2:1 ratio) of each primary variable. This presentation defined eight "sniff" combinations representing three levels of each derived variable. In an ANOVA of the logs of the responses, the effect of each primary variable was highly significant, with no significant interactions. A multiplicative regression model incorporating the effects of the three primary variables represented responses exceedingly well, with positive effects of N and T and a negative effect of V. When, with this model, the effect of each of the derived variables was isolated from the effects of all other variables, the analysis showed a positive effect for C, a near-zero positive effect for D, and a negative effect for F. Placing certain constraints upon the model parameters generates 13 distinct one- and two-variable models (e.g., the [C, T] model requires N and V to have equal but opposite effects). In ranking these reduced models in terms of their ability to predict the neural response, the predictive ability of [F, N] and [C, T] was at least as good as that of the three-variable model.     

Effects of antibodies against odorant binding proteins on electrophysiological responses to odorants

Monoclonal antibodies against two olfactory mucosal proteins, one with affinity for anisole-like and the other for benzaldehyde-like compounds, were applied to mouse olfactory epithelium. Responses to three odorants (anisole, benzaldehyde and amyl acetate) were measured. Of 26 antibodies, three (12%) inhibited responses only to the odorant with affinity for the antigen, nine (35%) inhibited responses to all three odorants, and 14 (54%) were without effect. None reduced responses by as much as 50%. The data support the hypothesis that there is a class of related proteins in olfactory neuronal cell membranes that function as receptor molecules and that other mechanisms also mediate odorant stimulation.     

Olfactory physiology in the Drosophila antenna and maxillary palp: acj6 distinguishes two classes of odorant pathways.

This article provides characterization of the electrical response to odorants in the Drosophila antenna and provides physiological evidence that a second organ, the maxillary palp, also has olfactory function in Drosophila. The acj6 mutation, previously isolated by virtue of defective olfactory behavior, affects olfactory physiology in the maxillary palp as well as in the antenna. Interestingly, abnormal chemosensory jump 6 (acj6) reduces response in the maxillary palp to all odorants tested except benzaldehyde (odor of almond), as if response to benzaldehyde is mediated through a different type of odorant pathway from the other odorants. In other experiments, different parts of the antenna are shown to differ with respect to odorant sensitivity. Evidence is also provided that antennal response to odorants varies with age, and that odorants differ in their age dependence.     

Honeybees Learn Odour Mixtures via a Selection of Key Odorants

Background

The honeybee has to detect, process and learn numerous complex odours from her natural environment on a daily basis. Most of these odours are floral scents, which are mixtures of dozens of different odorants. To date, it is still unclear how the bee brain unravels the complex information contained in scent mixtures.

Methodology/Principal Findings

This study investigates learning of complex odour mixtures in honeybees using a simple olfactory conditioning procedure, the Proboscis-Extension-Reflex (PER) paradigm. Restrained honeybees were trained to three scent mixtures composed of 14 floral odorants each, and then tested with the individual odorants of each mixture. Bees did not respond to all odorants of a mixture equally: They responded well to a selection of key odorants, which were unique for each of the three scent mixtures. Bees showed less or very little response to the other odorants of the mixtures. The bees'' response to mixtures composed of only the key odorants was as good as to the original mixtures of 14 odorants. A mixture composed of the other, non-key-odorants elicited a significantly lower response. Neither an odorant''s volatility or molecular structure, nor learning efficiencies for individual odorants affected whether an odorant became a key odorant for a particular mixture. Odorant concentration had a positive effect, with odorants at high concentration likely to become key odorants.

Conclusions/Significance

Our study suggests that the brain processes complex scent mixtures by predominantly learning information from selected key odorants. Our observations on key odorant learning lend significant support to previous work on olfactory learning and mixture processing in honeybees.     

Learning to smell the roses: experience-dependent neural plasticity in human piriform and orbitofrontal cortices

It is widely presumed that odor quality is a direct outcome of odorant structure, but human studies indicate that molecular knowledge of an odorant is not always sufficient to predict odor quality. Indeed, the same olfactory input may generate different odor percepts depending on prior learning and experience. Combining functional magnetic resonance imaging with an olfactory paradigm of perceptual learning, we examined how sensory experience modifies odor perception and odor quality coding in the human brain. Prolonged exposure to a target odorant enhanced perceptual differentiation for odorants related in odor quality or functional group, an effect that was paralleled by learning-induced response increases in piriform cortex and orbitofrontal cortex (OFC). Critically, the magnitude of OFC activation predicted subsequent improvement in behavioral differentiation. Our findings suggest that neural representations of odor quality can be rapidly updated through mere perceptual experience, a mechanism that may underlie the development of odor perception.     

Redundant neural circuits regulate olfactory integration

Olfactory integration is important for survival in a natural habitat. However, how the nervous system processes signals of two odorants present simultaneously to generate a coherent behavioral response is poorly understood. Here, we characterize circuit basis for a form of olfactory integration in Caenorhabditis elegans. We find that the presence of a repulsive odorant, 2-nonanone, that signals threat strongly blocks the attraction of other odorants, such as isoamyl alcohol (IAA) or benzaldehyde, that signal food. Using a forward genetic screen, we found that genes known to regulate the structure and function of sensory neurons, osm-5 and osm-1, played a critical role in the integration process. Loss of these genes mildly reduces the response to the repellent 2-nonanone and disrupts the integration effect. Restoring the function of OSM-5 in either AWB or ASH, two sensory neurons known to mediate 2-nonanone-evoked avoidance, is sufficient to rescue. Sensory neurons AWB and downstream interneurons AVA, AIB, RIM that play critical roles in olfactory sensorimotor response are able to process signals generated by 2-nonanone or IAA or the mixture of the two odorants and contribute to the integration. Thus, our results identify redundant neural circuits that regulate the robust effect of a repulsive odorant to block responses to attractive odorants and uncover the neuronal and cellular basis for this complex olfactory task.