Characterization of electro-olfactogram oscillations and their computational reconstruction

Electro-olfactogram (EOG) oscillations induced by odorant stimulation have been often reported in various vertebrates from fishes to mammals. However, the mechanism of generation of EOG oscillations remains unclear. In the present study, we first characterized the properties of EOG oscillations induced by amino acid odorants in the rainbow trout and then performed a computer simulation based on the main assumption that olfactory receptor neurons (ORNs) have intrinsic oscillatory properties due to two types of voltage-gated ion channels, which have not yet been reported in vertebrate ORNs. EOG oscillations appeared mostly on the peak and decay phases of negative EOG responses, when odorant stimuli at high intensity flowed regularly anterior to posterior olfactory lamellae in the olfactory organ. The appearance of EOG oscillations was dependent on the odorant intensity but not on the flow rate. The maximum amplitude and the maximum power frequency of EOG oscillations were 3.51 +/- 3.35 mV (mean +/- SD, n = 232, range 0.12-16.79 mV) and 10.59 +/- 5.05 Hz (mean +/- SD, n = 232, range 3.51-40.03 Hz), respectively. The simulation represented sufficiently well the characteristics of EOG oscillations; occurrence at high odorant concentration, odorant concentration-dependent amplitude and the maximum power frequency range actually observed. Our results suggest that EOG oscillations are due to the intrinsic oscillatory properties of individual ORNs, which have two novel types of voltage-gated ion channels (resonant and amplifying channels). The simulation program for Macintosh ('oscillation 3.2.4' for MacOS 8.6 or later) is available on the world wide web (http://bio2.sci.hokudai.ac.jp/bio/chinou1/noriyo_home.html).     

Neuropeptide Y Enhances Olfactory Mucosa Responses to Odorant in Hungry Rats

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

Ciliated olfactory receptor neurons in goldfish (Carassius auratus) partially survive nerve axotomy,rapidly regenerate and respond to amino acids

Electro-olfactogram (EOG) recordings in response to amino acid stimulation were made from both control and experimental olfactory mucosae following unilateral axotomy. The recorded EOG amplitudes, amino acid stimulus relative effectiveness and dose-response relations for control and experimental mucosae were comparable in all pre- and postoperative recordings. Semi-thin investigations of olfactory mucosae showed degeneration of olfactory receptors but indicated that intact receptors were also present. SEM of olfactory mucosae revealed that ciliated receptor cells were present in both axotomized and control sides on postoperative days, whereas microvillous receptors completely degenerated and did not regenerate until 7 weeks post axotomy. The present findings along with previous behavioral observations suggest at least three possible sources of the EOGs recorded from the experimental olfactory mucosae following olfactory nerve transection: (1) young olfactory receptor neurons whose axons had not yet reached the region of the transected olfactory nerve; (2) newly-emerged olfactory receptor neurons; and (3) olfactory receptor neurons that had not degenerated.Abbreviations EOG electro-olfactogram - SEM scanning electron microscopy (micrograph)     

Olfactory sensitivity to amino acids in the blackspot sea bream (<Emphasis Type="Italic">Pagellus bogaraveo</Emphasis>): a comparison between olfactory receptor recording techniques in seawater

The current study investigated the olfactory sensitivity of the blackspot sea bream to amino acids, odorants associated with food detection in fish, and compared the efficacy of two different experimental methods: multi-unit recording from the olfactory nerve and the electro-olfactogram (EOG). Twenty essential amino acids plus l-DOPA evoked clear, concentration-dependent olfactory responses using both methods, with estimated thresholds of 10^−8.5–10^−6.2 M (nerve recording) and 10^−7.5–10^−4.8 M (EOG). The most potent amino acids were l-cysteine, l-methionine (both sulphur-containing), l-alanine, l-leucine (both neutral), l-glutamine (amide-containing) and l-serine (hydroxyl-containing). The least potent were l-proline (secondary α-amino group), the aromatic amino acids and glycine (simplest). Although the rank order of olfactory potency was similar for the two methods used, and the calculated thresholds given by the two methods were positively correlated, the sensitivity of the EOG was consistently lower than multi-unit recording by approximately one order of magnitude, presumably due to the electrical shunting effect of seawater. As in freshwater, the EOG could be a valid method for comparing olfactory potency of different odorants in stenohaline marine fish; however, for absolute ‘biological’ thresholds, a more invasive recording technique, such as multi-unit recording from the olfactory nerve, should be used.     

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.     

Onset of odorant receptor gene expression during olfactory sensory neuron regeneration

Individual olfactory sensory neurons are thought to express only one odorant receptor gene from a repertoire of hundreds to thousands of genes. How do these sensory neurons choose just one specific odorant receptor to express during their differentiation? As an initial attempt toward understanding the process of odorant receptor gene regulation, we studied when odorant receptor expression is activated during sensory neuron regeneration. We find that receptor gene expression is activated in postmitotic neurons and can occur in the absence of the olfactory bulb. These results suggest that receptor expression is restricted to the terminal stages of olfactory neuron differentiation, and sensory neurons do not simply inherit the odorant receptor that is already expressed in mitotic precursor cells. Our results also support a model in which odorant receptor gene expression occurs independent of the olfactory bulb.     

Effects of concentration and sniff flow rate on the rat electroolfactogram

Previous reports using the electroolfactogram (EOG) to study the spatial and temporal aspects of response in the rodent olfactory epithelium had focused on high odorant concentrations that gave large responses. This investigation has used lower concentrations to test the difference between responses in the rat dorsomedial and lateral recesses with a range of nasal flow rates and a range of chemical properties. The responses to a highly polar, more hydrophilic odorant changed more steeply with flow rate than responses to a very nonpolar, hydrophobic odorant. With low flow rates there was a response delay in the lateral recess, which is consistent with the models indicating lower flow rates in that region. We observed significant volume conduction effects in which large responses in the dorsomedial region obscured smaller initial portions of the lateral responses. These effects could be removed by destroying the dorsomedial response with a high concentration of a low molecular weight ester. We caution that investigators of EOG recordings from the intact epithelium must attend to the possible presence of volume conduction, which can be assessed by attention to the selectivity of odorant response, response waveform, and response latency.     

Transient and persistent tetrodotoxin-sensitive sodium currents in squid olfactory receptor neurons

Squid olfactory receptor neurons are primary bipolar sensory neurons capable of transducing water-born odorant signals into electrical impulses that are transmitted to the brain. In this study, we have identified and characterized the macroscopic properties of voltage-gated Na⁺ channels in olfactory receptor neurons from the squid Lolliguncula brevis. Using whole-cell voltage-clamp techniques, we found that the voltage-gated Na⁺ channels were tetrodotoxin sensitive and had current densities ranging from 5 to 169 pA pF⁻¹. Analyses of the voltage dependence and kinetics revealed interesting differences from voltage-gated Na⁺ channels in olfactory receptor neurons from other species; the voltage of half-inactivation was shifted to the right and the voltage of half-activation was shifted to the left such that a “window-current” occurred, where 10–18% of the Na⁺ channels activated and did not inactivate at potentials near action potential threshold. Our findings suggest that in squid olfactory neurons, a subset of voltage-gated Na⁺ channels may play a role in generating a pacemaker-type current for setting the tonic levels of electrical activity required for transmission of hyperpolarizing odor responses to the brain. Accepted: 1 October 1998     

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.     

Inhibition of protein synthesis does not block physiological responses to odors in the frog over the short term

Frog olfactory mucosae were stimulated with an odorant every 60 sec and electrophysiological responses (electroolfactograms or EOGs) were monitored continuously--before, during, and after treatment with puromycin, a protein synthesis inhibitor. Repeated administration of puromycin at 4 hr intervals over a 36 hr period failed to suppress EOG responses. In an alternative approach, olfactory responses were inhibited with ethyl bromoacetate, a vaporous alkylating agent. EOG responses failed to reappear over a 72 hr period. We conclude that receptor turnover is not readily influenced by stimulation, and that the turnover time of frog olfactory receptor proteins is of the order of days or longer.     

Multiple axon guidance cues establish the olfactory topographic map: how do these cues interact?

Each primary olfactory neuron stochastically expresses one of approximately 1000 odorant receptors. The total population of these neurons therefore consists of approximately 1,000 distinct subpopulations, each of which are mosaically dispersed throughout one of four semi-annular zones in the nasal cavity. The axons of these different subpopulations are initially intermingled within the olfactory nerve. However, upon reaching the olfactory bulb, they sort out and converge so that axons expressing the same odorant receptor typically target one or two glomeruli. The spatial location of each of these approximately 1800 glomeruli are topographically-fixed in the olfactory bulb and are invariant from animal to animal. Thus, while odorant receptors are expressed mosaically by neurons throughout the olfactory neuroepithelium their axons sort out, converge and target the same glomerulus within the olfactory bulb. How is such precise and reproducible topographic targeting generated? While some of the mechanisms governing the growth cone guidance of olfactory sensory neurons are understood, the cues responsible for homing axons to their target site remain elusive.     

Electroolfactogram responses from organotypic cultures of the olfactory epithelium from postnatal mice

Organotypic cultures of the mouse olfactory epithelium connected to the olfactory bulb were obtained with the roller tube technique from postnatal mice aged between 13 and 66 days. To test the functionality of the cultures, we measured electroolfactograms (EOGs) at different days in vitro (DIV), up to 7 DIV, and we compared them with EOGs from identical acute preparations (0 DIV). Average amplitudes of EOG responses to 2 mixtures of various odorants at concentrations of 1 mM or 100 microM decreased in cultures between 2 and 5 DIV compared with 0 DIV. The percentage of responsive cultures was 57%. We also used the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to trigger the olfactory transduction cascade bypassing odorant receptor activation. Average amplitudes of EOG responses to 500 microM IBMX were not significantly different in cultures up to 6 DIV or 0 DIV, and the average percentage of responsive cultures between 2 and 5 DIV was 72%. The dose-response curve to IBMX measured in cultures up to 7 DIV was similar to that at 0 DIV. Moreover, the percentage of EOG response to IBMX blocked by niflumic acid, a blocker of Ca-activated Cl channels, was not significantly different in cultured or acute preparations.     

Whole-cell recordings and photolysis of caged compounds in olfactory sensory neurons isolated from the mouse

Gene manipulation and molecular biological techniques for the study of olfaction are well developed in mice, while electrophysiological properties of mouse olfactory sensory neurons have been less extensively investigated. We used the whole-cell voltage-clamp technique in mouse isolated olfactory sensory neurons to investigate both voltage-gated and transduction currents. Voltage-gated currents were composed of transient inward currents followed by outward currents with transient and sustained components. Of the tested olfactory sensory neurons, 12% responded to the odorant cineole with an inward current. Caged compounds were introduced into the cytoplasm through the patch pipette and flash photolysis of caged cyclic nucleotides activated an inward current in 94% of the cells. When the flash was localized at the cilia, the response latency, rising time and duration were shorter than when the flash illuminated the soma. The amplitude of the photolysis response was dependent on light intensity and the relation was fitted by the Hill equation, with a Hill coefficient of 3.2. These results demonstrate that it is possible to obtain recordings in the whole-cell configuration from olfactory sensory neurons isolated from the mouse and that voltage-gated currents and transduction properties are largely similar to those of amphibians.     

Odor suppression of voltage-gated currents contributes to the odor-induced response in olfactory neurons

Olfactorychemotransduction involves a signaling cascade. In addition totriggering transduction, odors suppress ion conductances. Bystimulating with brief odorant pulses, we observed a current associatedwith odor-induced suppression of voltage-gated conductances and studiedits time dependence. We characterized this suppression current inisolated Caudiverbera caudiverberaolfactory neurons. All four voltage-gated currents are suppressed byodor pulses in almost every neuron, and suppression is caused by odorsinducing excitation and by those inducing inhibition, indicating anonselective phenomenon, in contrast to transduction. Suppression has a10-fold shorter latency than transduction. Suppression was morepronounced when odors were applied to the soma than to the cilia,opposite to transduction. Suppression was also present in rat olfactory neurons. Furthermore, we could induce it inDrosophila photoreceptor cells,demonstrating its independence from the chemotransduction cascade. Weshow that odor concentrations causing suppression are similar to thosetriggering chemotransduction and that both suppression and transductioncontribute to the odor response in isolated olfactory neurons.Furthermore, suppression affects spiking, implying a possiblephysiological role in olfaction.

     

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.     

Effect of antibody to anisole binding protein on odorant perturbation of Na+-K+ ATPase activity

Odorant perturbation of Na⁺-K⁺ ATPase activity from cow olfactorytissue was strongly affected by ng quantities of antibodiesto anisole binding protein from dog olfactory mucosa. Antibodyprotein (80 ng per ml reaction mixture) prevented odorant perturbationof Na⁺-K⁺ ATPase activity. Antibody effect on odorant perturbationshowed concentration dependence and was active against a numberof different odorous chemicals. Electrophysiological studies(Goldberg et al, 1979) showed that mouse EOG responses due toodorants were inhibited 50% by previous exposure to 0.8 ng antibodies.Thus electrophysiological and biochemical responses showed sensitivityto the antibodies from the anisole binding protein from dogolfactory tissue. It is proposed that NA⁺-K⁺ ATPase may participatein the initiation of nerve signals caused by odorant-enzymecomplex interactions.     

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.     

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.     

Joint migration of gonadotropin-releasing hormone (GnRH) and neuropeptide Y (NPY) neurons from olfactory placode to central nervous system

The olfactory epithelium in vertebrates generates the olfactory sensory neurons and several migratory cell types. Prominent among the latter are the gonadotropin-releasing hormone (GnRH) neurons that differentiate within the olfactory epithelium during embryogenesis and migrate along the olfactory nerve to the central nervous system. We initiated studies to characterize additional neuronal phenotypes of olfactory epithelial derivation. Neuropeptide Y (NPY) neurons are functionally related to the reproductive axis, modulating the release of GnRH and directly enhancing GnRH-induced luteinizing hormone (LH) secretion from gonadotrophs. We demonstrate that a population of migratory NPY neurons originates within the olfactory epithelium of the chick. At stage 25, NPY-positive fibers, but not cells, were detected in the epithelium and the nerve. By stages 28–34, NPY neurons and processes were present in the olfactory epithelium, olfactory nerve, and at the junction of the olfactory nerve and forebrain. In these regions the number of NPY neurons increased until stage 30 and then declined as development progressed. Electron microscopic immunocytochemistry confirmed the neuronal phenotype of the NPY-positive cells. The origin and migratory nature of some of these NPY cells was confirmed by double-label immunocytochemical detection of NPY and GnRH. A large percentage of the NPY-cells coexpressed the GnRH peptide. Between stages 28 and 34 single- and double-labeled NPY and GnRH neurons were found side by side along the GnRH migratory route emanating from the nasal epithelium, along the olfactory nerve, and into the ventral forebrain. These data suggest that an NPY population originates in the olfactory epithelium and migrates into the central nervous system during embryogenesis. By stage 42, no NPY/GnRH double-labeled cells were detected. © 1996 John Wiley & Sons, Inc.